Ernö Zádor

Intermittent spontaneous breathing protects the rat diaphragm from mechanical ventilation effects

Objective:Short-term mechanical ventilation has been proven to reduce diaphragm force and fiber dimensions. We hypothesized that intermittent spontaneous breathing during the course of mechanical ventilation would minimize the effects of mechanical ventilation on diaphragm force and expression levels of transcription factors (MyoD and myogenin). Design:Randomized, controlled experiment. Setting:Animal basic science laboratory. Subjects:Male Wistar rats, weighing 350–500 g. Interventions:Anesthetized and tracheotomized rats were submitted to either 24 hrs of spontaneous breathing (SB, n = 5), 24 hrs of continuous controlled mechanical ventilation (CMV, n = 7), or controlled mechanical ventilation with intermittent spontaneous breathing: 60 mins every 5 hrs of mechanical ventilation repeated four times (ISB60, n = 8) or 5 mins every 5 hrs 55 mins of mechanical ventilation repeated four times (SB5, n = 9). They were compared with control animals free from intervention (C, n = 5). Measurements and Main Results:The profile of the diaphragm force-frequency curve of the controls and SB group was significantly different from that of the ISB and CMV groups; especially, the mean asymptotic force was less in the ISB and CMV compared with controls and SB. CMV resulted in a significant decrease in the diaphragm type I (−26%, p < .05 vs. C) and type IIx/b (−39%, p < .005 vs. C and SB) cross-sectional area, whereas this was not observed in the ISB groups. Diaphragm MyoD protein expression was significantly decreased after ISB60 (−35%, p < .0001 vs. C and SB) and even more after CMV (−73%, p < .0001 vs. others). The same pattern was observed with myogenin protein levels. Positive relationships between diaphragm MyoD and myogenin protein levels and diaphragm force were observed. Conclusions:The data demonstrated that intermittent spontaneous breathing during the course of mechanical ventilation may minimize the deleterious effect of controlled mechanical ventilation on diaphragm force, fiber dimensions, and expression of transcription factors.

Sarcolipin and phospholamban mRNA and protein expression in cardiac and skeletal muscle of different species

The widely held view that SLN (sarcolipin) would be the natural inhibitor of SERCA1 (sarcoplasmic/endoplasmic-reticulum Ca2+-ATPase 1), and PLB (phospholamban) its counterpart for SERCA2 inhibition is oversimplified and partially wrong. The expression of SLN and PLB mRNA and protein relative to SERCA1 or SERCA2 was assessed in ventricle, atrium, soleus and EDL (extensor digitorum longus) of mouse, rat, rabbit and pig. SLN protein levels were quantified by means of Western blotting using what appears to be the first successfully generated antibody directed against SLN. Our data confirm the co-expression of PLB and SERCA2a in cardiac muscle and the very low levels (in pig and rabbit) or the absence (in rat and mouse) of PLB protein in the slow skeletal muscle. In larger animals, the SLN mRNA and protein expression in the soleus and EDL correlates with SERCA1a expression, but, in rodents, SLN mRNA and protein show the highest abundance in the atria, which are devoid of SERCA1. In the rodent atria, SLN could therefore potentially interact with PLB and SERCA2a. No SLN was found in the ventricles of the different species studied, and there was no compensatory SLN up-regulation for the loss of PLB in PLB(-/-) mouse. In addition, we found that SLN expression was down-regulated at the mRNA and protein level in the atria of hypertrophic hearts of SERCA2(b/b) mice. These data suggest that superinhibition of SERCA by PLB-SLN complexes could occur in the atria of the smaller rodents, but not in those of larger animals.

Phytoecdysteroids and anabolic-androgenic steroids--structure and effects on humans.

Phytoecdysteroids are structural analogs of the insect molting hormone ecdysone. Plants comprise rich sources of ecdysteroids in high concentration and with broad structural diversity. Ecdysteroids have a number of proven beneficial effects on mammals but the hormonal effects of ecdysteroids have been proven only in arthropods. Their structures are somewhat similar to those of the vertebrate steroid hormones but there are several structural differences between the two steroid groups. Despite of these essential structural differences, ecdysteroids exert numerous effects in vertebrates that are similar to those of vertebrate hormonal steroids, and they may serve as effective anabolic, hepatoprotective, immunoprotective, antioxidant and hypoglycemic agents. Ecdysteroids do not bind to the cytosolic steroid receptors, instead, they are likely to influence signal transduction pathways, like the anabolic steroids, possibly via membrane bound receptors. The application of phytoecdysteroids is a promising alternative to the use of anabolic-androgenic steroids because of the apparent lack of adverse effects. The prospective use of phytoecdysteroids may extend to treatments of pathological conditions where anabolic steroids are routinely applied. One of the most cited aspects of phytoecdysteroid application (on the Internet) is the increase of muscle size. However in this field too stringent research is needed as an adequate cytological explanation is not yet available for the anabolic. This paper reports on the most important structural differences between androgenic hormones, their synthetic analogs and ecdysteroids. The anabolic/hormonal effects and the possible mechanisms of action of these compounds are also discussed as concerns the skeletal muscle.

Regenerating soleus and extensor digitorum longus muscles of the rat show elevated levels of TNF-α and its receptors, TNFR-60 and TNFR-80

We measured the mRNA and protein levels of tumor necrosis factor‐α (TNF‐α) and the transcript levels of its receptors (TNFR‐60 and TNFR‐80) in the rat soleus (slow twitch) and extensor digitorum longus (EDL; fast twitch) muscles regenerating from notexin‐induced necrosis. On the first day after administration of the toxin, when most fibers were necrotic and invaded by inflammatory cells/macrophages, dramatic increases of transcript and protein levels of TNF‐α and of the mRNA levels of its receptors were observed. The transcript levels of TNF‐α and TNFR‐60, but not of TNFR‐80, showed a second but smaller increase at the time when newly formed muscle fibers became reinnervated. In situ hybridization showed that on day 1, during the phase of extensive necrosis, the transcript of TNF‐α was abundantly present and on day 4 of regeneration it was most often seen in areas devoid of desmin. The mRNA level of TNF‐α was not detectable in BC3H1‐ and C2C12‐cultured myoblasts and it was low in freeze‐injured muscle, corresponding to the relatively mild degree of inflammation elicited by freezing. Therefore, our results are most consistent with the view that inflammatory cells/macrophages are the main source of TNF‐α.

Myostatin levels in regenerating rat muscles and in myogenic cell cultures.

Myostatin is a newly described member of the TGF-β superfamily acting as a secreted negative regulator of skeletal muscle mass in several species, but whose mode of action remains largely unknown. In the present work, we followed the myostatin mRNA and protein levels in rat soleus and extensor digitorum longus (EDL) muscles regenerating in vivo from notexin-induced necrosis, and the myostatin transcript levels in two different in vitro myogenic differentiation models: i.e. in mouse BC3H1 and C2C12 cultured cells. The in vivo regenerating rat skeletal muscles showed a characteristic time-dependent expression of myostatin mRNA. After notexin injection, the transcript levels dropped below the detection limit on day 1 in soleus and close to the detection limit on day 3 in EDL, then increased to a maximum on day 7 in soleus and after 28 days finally reached the control values in both types of muscles. In contrast, the myostatin protein levels increased dramatically on the first days of regeneration in both muscles, i.e. at the time when its transcript level was low. Later on the myostatin protein level gradually declined to normal in soleus while in EDL it stayed high some days longer and decreased to normal on days 21–28. In vitro proliferating myoblasts produced low level of myostatin mRNA, which increased upon induction of differentiation suggesting that functional innervation is no prerequisite for myostatin expression. Myostatin production in vitro seems not to be dependent on myocyte fusion either, since it is observed in differentiated BC3H1 cells, which are defective in myofiber formation.

20-Hydroxyecdysone increases fiber size in a muscle-specific fashion in rat

20-Hydroxyecdysone (20E) is an ecdysteroid hormone that regulates moulting in insects. Interestingly, 20E is also found most abundantly in plant species and has anabolic effects in vertebrates, i.e. increasing muscle size without androgen influence. The effect of 20E on slow and fast fiber types of skeletal muscle has not been reported yet. Here we present that 20E affects the size (cross-sectional area, CSA) of the different fiber types in a muscle-specific manner. The effect on fiber size was modified by the distance from the site of the treatment and the presence of a regenerating soleus muscle in the animal. Besides the fiber size, 20E also increased the myonuclear number in the fibers of normal and regenerating muscles, suggesting the activation of satellite cells. According to our results 20E may provide an alternative for substitution of anabolic-androgenic steroids in therapeutic treatments against muscle atrophy.

Prolonged passive stretch of rat soleus muscle provokes an increase in the mRNA levels of the muscle regulatory factors distributed along the entire length of the fibers

The mRNA levels of the adult and the neonatal sarcoplasmic/endoplasmic reticulum Ca2+-ATPases (SERCA1a and SERCA1b, respectively) and those of the muscle regulatory factors (MRFs: myoD, myf-5, myogenin, MRF4) have been assessed by RT PCR in rat soleus muscles immobilized for 3 days in an extended position (passive stretch). The transcript level of the fast type SERCA1a Ca2+-transport ATPase decreased to half of its normal value, whereas that of neonatal SERCA1b isoform increased 5-fold above control in stretched muscles. Immunostaining of muscle cross sections showed that the fraction of fibers expressing the SERCA1a protein was decreased evenly along the length of the stretched muscles indicating that a transformation occurred of fast fibers to slow ones. The mRNA levels of MRFs were elevated 3- to 6-fold above the normal level and were distributed evenly along the length of the stretched muscles. However in the controls these transcripts were more abundant at both ends of the muscle. The stretch increased the level of myoD and immunocytochemistry showed the expression of myoD protein in a number of nuclei of the stretched muscles whereas it was practically undetectable by this method in the control muscles. Western blotting did not indicate a significant stretch-induced increase in the level of the myogenin protein, in spite of the fact that immunocytochemistry tended to show more myogenin-positive nuclei in stretched muscles as compared to the controls. These data indicate that after 3 days of passive stretch the central and the terminal parts of the soleus muscle adapt similarly by increasing the levels of the MRFs, by decreasing the overall levels of the fast SERCA1-type of ATPase and by partially re-establishing a neonatal mode of alternative SERCA1 transcript splicing resulting in an increased SERCA1b/1a ratio.

Expression of sarcoplasmic/endoplasmic reticulum Ca2+ ATPases in the rat extensor digitorum longus (EDL) muscle regenerating from notexin-induced necrosis.

The level of sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA) mRNAs and proteins have been assessed by␣RT-PCR,␣immunoblotting and immunocytochemistry in the rat extensor digitorum longus (EDL) muscles during regeneration from notexin-induced necrosis. As a result of the necrosis, SERCA1 and SERCA2 declined on days 1 and 3 after␣administration of the toxin. Thereupon the mRNA of the fast isoform SERCA1 rapidly increased between days 5 and 10 to the normal level. The mRNA level of the “housekeeping” SERCA2b isoform increased markedly during the actual necrosis at days 1 and 5, probably due to invading cells. Then the mRNA level of the neonatal SERCA1b splice variant increased first, and exceeded the level of the adult SERCA1a transcript on day 5. At later stages of regeneration the neonatal form was gradually replaced by the adult SERCA1a form, thus recapitulating similar changes known to occur during normal ontogenesis. Along with S ERCA1, the levels of the slow isoform (SERCA2a) mRNA and protein increased on day 5, but the␣SERCA2a mRNA levels never rose above 10% of SERCA1 and after 10 days gradually declined again. In the normal and␣regenerated muscles, SERCA1 was expressed in 97% of the fibres which accounted for the population of fast-twitch fibres␣(type IIa, type IIb and probably type IIx/d). SERCA2a was present in 6% of the fibres of normal muscle (mostly in the␣slow-twitch type I fibres). At the end of regeneration the number of fibres expressing SERCA2a was twice as high and␣were␣found in small groups, unlike in normal EDL, but about 50% of these clustered fibres also expressed SERCA1.

Phytoecdysteroids and Vitamin D Analogues - Similarities in Structure and Mode of Action

Phytoecdysteroids are plant steroids with identical or analogue structures to the molting hormone in arthropods. The ecdysteroids exert several beneficial effects on mammals, from which the most cited and deeply examined one is the increase of muscle size and strength. This shows similarities with the mode of action of the androgenic steroids but the ecdysteroids do not bind to the cytoplasmic/nuclear receptor of the mammalian steroids. These findings led to the hypothesis that ecdysteroids possibly bind to membrane bound receptors and they are likely to influence signal transduction pathways. Probably because of their closely related chemical structures, ecdysteroids exert some similar effects in vertebrates to those of the hormone 1 alpha,25-dihydroxyvitamin D3 (1,25D) which is produced in the kidney from 25-hydroxyvitamin D3, after being converted in the liver from Vitamin D3. 1,25D generates biological responses via both genomic and rapid, nongenomic mechanisms. Structure-activity relationship studies with different Vitamin D analogues could open the possibility to show that the two ways of action (genomic and nongenomic) can be influenced separately. The connection between the Vitamin D status and muscle function is already well-described in clinical studies, and several efforts have been made to evaluate the effect of Vitamin D deficiency or supplementation on muscle morphological changes and the underlying molecular mechanisms. This paper aims to summarize the main structural commonalities between the ecdysteroids, 1,25D and other Vitamin D analogues. The similarities in their effects and pathways that might be involved in the mechanism of action of these compounds will also be discussed.

The Compact mutation of myostatin causes a glycolytic shift in the phenotype of fast skeletal muscles

Myostatin is an important negative regulator of skeletal muscle growth. The hypermuscular Compact (Cmpt) mice carry a 12-bp natural mutation in the myostatin propeptide, with additional modifier genes being responsible for the phenotype. Muscle cellularity of the fast-type tibialis anterior (TA) and extensor digitorum longus (EDL) as well as the mixed-type soleus (SOL) muscles of Cmpt and wild-type mice was examined by immunohistochemical staining of the myosin heavy chain (MHC) proteins. In addition, transcript levels of MHC isoforms were quantified by qPCR. Based on our results, all investigated muscles of Cmpt mice were significantly larger compared with that of wild-type mice, as characterized by fiber hyperplasia of different grades. Fiber hypertrophy was not present in TA; however, EDL muscles showed specific IIB fiber hypertrophy while the (I and IIA) fibers of SOL muscles were generally hypertrophied. Both the fast TA and EDL muscles of Cmpt mice contained significantly more glycolytic IIB fibers accompanied by a decreased number of IIX and IIA fibers; however, this was not the case for SOL muscles. In summary, despite the variances found in muscle cellularity between the different myostatin mutant mice, similar glycolytic shifts were observed in Cmpt fast muscles as in muscles from myostatin knockout mice.