Satu Mänttäri

Effects of different training protocols on Ca2+ handling and oxidative capacity in skeletal muscle of Atlantic salmon (Salmo salar L.)

SUMMARY The modulation of calcium channel density and oxidative capacity in skeletal muscle after different training protocols were studied in 3-year-old Atlantic salmon smolts. The effect of endurance exercise on dihydropyridine (DHP) and ryanodine (Ry) receptor densities as well as on muscle metabolism were determined by immunoblot and histochemical analysis from swimming muscles of fish subjected to nine different training protocols varying in duration and water current velocity. In general, exercise training caused a significant increase in the density of both DHP and Ry receptors in both muscle types studied. In red muscle, the most notable increase in DHP and Ry receptor expression was observed in muscle sections from fish swimming against intermediate current velocity for a 2-week period (182.3±16.3%, 234.6±30.3%, respectively). In white muscle, the expression of DHP and Ry receptors was most upregulated after a 6-week swimming period also at intermediate water current velocity (270.4±23.9%, 114.4±15.3%, respectively). As with the activity of enzymes involved in muscle energy supply, endurance exercise resulted in a significant increase in succinate dehydrogenase (SDH) activity, but a significant decrease in phosphorylase activity. We conclude that the expression of both DHP and Ry receptors was upregulated in the swimming muscles of salmon as a consequence of exercise training. This, along with the increased oxidative enzyme activity, provides benefits to the contraction efficiency of fish muscles while swimming. However, it was also observed that optimal oxidative swimming capacity is achieved only with a proper exercise program, since the most relevant changes in DHP and Ry receptor expression, as well as in oxidative capacity, were seen in the group training with the intermediate swimming velocity.

The effect of myocardial infarction on the synthesis, concentration and receptor expression of endogenous melatonin.

Abstract:  We examined the time course of changes in the synthesis and levels of endogenous melatonin and in the expression of MT1 and MT2 melatonin receptors 1 day, 2 and 4  wk after myocardial infarction (MI) in rats. MI was produced by ligation of the left anterior descending coronary artery. Transthoracic echocardiography was performed to characterize structural and functional changes after MI. mRNA levels were measured by real‐time quantitative reverse transcription‐polymerase chain reaction and proteins by Western blotting. One day after infarction, MI rats had 4.3 times (P < 0.001) higher pineal melatonin synthesis, than sham‐operated animals, which was associated with the increased concentration of melatonin in plasma (P < 0.001) and left ventricle (LV) (P = 0.01). The amount of MT1 receptor protein decreased significantly in MI LVs compared with control LVs 1 day after infarction (P < 0.01), followed by recovery during the next 2 wk. Furthermore, the expression of MT1 receptor mRNA of the MI LVs was elevated 2 wk after infarction (P < 0.01) compared with control LVs. The amount of MT2 receptor proteins in MI LVs was higher than in sham‐operated LVs 1 day (P < 0.05) and 4 wk (P < 0.01) after MI. In conclusion, melatonin synthesis in the pineal gland increased rapidly in response to the MI, supporting an important role for endogenous melatonin in protecting the heart after MI. The observed changes in the expression of MT1 and MT2 receptors suggest that melatonin receptors may be involved in mediating, at least, in part, the protective effects of melatonin in the heart after infarction.

Comparative analysis of mouse skeletal muscle fibre type composition and contractile responses to calcium channel blocker

BackgroundIn this study, we examined the correlation between excitation-contraction coupling characteristics and skeletal muscle fibre type by (1) localizing the distribution of dihydropyridine receptor (DHPR) protein and (2) comparing the effect of DHPR blocker on muscles with different fibre type composition, in order to better understand the differences between contractile phenotypes of fibres and to explain the contradictory reports to date on the interaction of dihydropyridines with skeletal muscle isoform of DHPR.ResultsHistochemical experiments revealed that fluorophore conjugated dihydropyridines stain selectively the membranes of muscle fibres. The staining was most evident in type IIA fibres. The major fibre type in gluteus and femoris, revealed by mATPase staining, was IIA (45.0 and 38.1 %, respectively). In gastrocnemius the content of IIA fibres was 22.7 %. Contraction forces before and after the addition of blocker for the three muscles investigated were: gluteus 0.075 ± 0.017 N vs. 0.052 ± 0.011 N, femoris 0.045 ± 0.005 N vs. 0.033 ± 0.005 N and gastrocnemius 0.089 ± 0.016 N vs. 0.075 ± 0.014 N, respectively. The attenuation of contraction force proportional to the cross-sectional area of the muscle was significantly (P = 0.023) higher in gluteus (28.3 ± 3.5 %) and femoris (27.6 ± 3.2 %) as compared to gastrocnemius (16.1 ± 2.5 %). However, no significant change in the control measurements was observed ruling out the possibility of fatigue.ConclusionThe results indicate that the attenuation of the contraction force was largest in muscles with a high percentage of type IIA fibres. This supports our finding that the abundance of dihydropyridine receptors of IIA fibres outnumbers that in the other fibre types. The present data show that the correlation of density of dihydropyridine receptors can be one of the important factors influencing the overall contractile properties of the muscle and for its part explain the contradictory results of previous studies on coupling process.

Encephalopsin (OPN3) protein abundance in the adult mouse brain

Encephalopsin belongs to the family of extraretinal opsins having a putative role in CNS tissue photosensitivity. Encephalopsin mRNA has earlier been localized in rodent brains, but expression and localization of the protein has not yet been reported. In this study, we aimed to define encephalopsin protein abundance and localization in the rodent brain. The distribution and localization of encephalopsin protein in a mouse brain and selected peripheral tissues were analysed in ten mice, using Western blotting and immunohistochemistry. The specificity of immunoreaction was validated by primary antibody omitting and immunizing peptide blocking experiment. We found encephalopsin protein abundant in the mouse brain, but not in the periphery. Encephalopsin protein was present in neurons of the mouse cerebral cortex, paraventricular area, and cerebellar cells. Our results show that encephalopsin is expressed at the protein level in different brain areas of the mouse. Therefore, the suggested idea that encephalopsin plays a role in non-visual photic processes seems to be applicable. Evidently, further investigations are needed to find out the signalling mechanisms, and the potential physiological role of encephalopsin in phototransduction due to the changes in ambient light.

Immunohistochemical definition of MT2 receptors and melatonin in the gastrointestinal tissues of rat

The gastrointestinal tract of vertebrate species contains melatonin, which participates in several physiological functions. Some of these effects are mediated via specific membrane receptors (MT(1) and MT(2)). In the present study, the distribution of the MT(2) receptor protein in the gastrointestinal tract was localized, and changes in MT(2) receptor density were observed in relation to the expected circadian changes in melatonin concentrations. Immunohistochemistry was performed in the rat stomach, duodenum, colon and pancreas. The amount of MT(2) was determined by Western blot. Melatonin concentrations were measured by radioimmunoassay (RIA). In the stomach, duodenum and colon, the most intense immunoreactivity was observed in the Muscularis mucosae and in the Muscularis externa. In the pancreas, the immunolabelling was less intense. There was a clear daily rhythm of melatonin concentrations in the stomach, duodenum and pancreas, with higher levels during the dark period. The density of MT(2) receptors did not exhibit circadian variation. Moreover, circadian changes in melatonin concentrations were not found in the colon. The density of MT(2) was the highest in the colon. Our results provide evidence for the heterogeneous distribution of MT(2) receptors in different parts and layers of the gastrointestinal tract, which could indicate a physiological role of melatonin in the gastrointestinal tract.

Effects of training on functional variables of muscles in reared Atlantic salmon Salmo salar smolts : connection to downstream migration pattern

The relative amount of muscle contraction regulating dihydropyridine and ryanodine receptors in the swimming muscles of trained reared Atlantic salmon Salmo salar smolts was compared with those of untrained and wild smolts. After an optimized 2 week training period, i.e. swimming with a velocity of 1·5 body lengths per second for 6 h per day, the level of both receptors was significantly higher in the muscles of trained S. salar than in the untrained ones before they were released into the natural environment. This difference persisted after downstream migration in the river. The highest level of receptors was observed in wild S. salar. Swimming performance was also higher in trained fish compared to untrained ones. Furthermore, swimming performance was positively associated with the level of receptors in both red and white muscle types. Downstream migration after release into the wild was significantly slower in trained smolts than in untrained fish. This indicates that trained smolts were most probably swimming harder against the current in the river than untrained smolts. The possible advantages for a slower migration in the river are discussed. This study shows that the prerequisites for effective contraction of the swimming muscles are better met in trained S. salar compared to untrained fish, and the muscles of trained smolts more closely resemble those of wild smolts. The results also imply that the capacity of untrained, reared smolts to swim against the current is not equal to that of their trained or wild counterparts which affects the downstream migration pattern of S. salar smolts.

Expression of L-type calcium channels associated with postnatal development of skeletal muscle function in mouse

Several factors have an influence on the improvement of muscle activity and motor co-ordination of mammals during post-natal development. One of them is voltage sensitive L-type calcium channel function. In striated muscles of adult mammals these channels are located in T-tubule membranes thus linking the on-coming action potential to the molecular process of muscle contraction. The postnatal development of L-type calcium channels is therefore critical not only for contraction but also for all subsequent motor learning. We used high affinity enantiomer of dihydropyridine labelled with a fluorophore in order to show the relative amount of L-type calcium channels by histofluorescence in tissue. We found by qualitative microscopical analysis that the amount of L-type calcium channels increased during the postnatal development in the mouse skeletal muscle (m. rectus femoris and m. gastrocnemius). We also noted variation between different fibre types in the increase of the amount of L-type calcium channels, as judged by the intensity of histofluorescence. We showed by histochemical staining and statistical analysis that the high density of L-type calcium channels in adult muscles is correlated with fast oxidative glycolytic fibre type of striated muscles rather than slow oxidative or fast glycolytic fibres. Based on this finding we propose that the development of L-type calcium channels can be considered as one of the factors determining the different physiological properties of fibre types.

Egg size and composition in an ageing capital breeder – consequences for offspring performance

1. Egg size is often used as a proxy of egg quality although size and composition may vary, e.g. in insects egg size usually decreases as female ages. Whether this decrease in size reflects reduced concentrations of essential nutrients such as lipids and proteins of eggs laid by ageing females, or does reduced size per se explain often observed lower fitness of later laid eggs is poorly explored.

Effects of low-intensity training on dihydropyridine and ryanodine receptor content in skeletal muscle of mouse

To evaluate low-intensity exercise training induced changes in the expression of dihydropyridine (DHP) and ryanodine (Ry) receptors both mRNA and protein levels were determined by quantitative RT-PCR and immunoblot analysis from gastrocnemius (GAS) and rectus femoris (RF) muscles of mice subjected to a 15-week aerobic exercise program. The level of muscular work was assayed by changes in myosin heavy chain (MHC) content, myoglobin (Mb) expression and muscle size. The mRNA expression and optical density of DHP receptor increased significantly in GAS by 66.8 and 39.5%, respectively. The expression of Ry receptor, on the other hand, was not up-regulated. In RF, there was a significant increase of 38.4% in the mRNA expression of DHP receptor, although the protein level remained the same. No changes in Ry receptor expression was observed. The training resulted in a 1.58% increase in the amount of MHC IIa and a 2.34% decrease in that of IIb and IId in GAS. A significant 8.3% increase in the Mb content was observed. In RF, no significant changes in MHC or in Mb content were noted. Our results show that an evident increase in the mRNA and protein expression of DHP receptor was induced in GAS even by a relatively low-intensity exercise. Surprisingly, contrast to DHP receptor expression, no changes in Ry receptor mRNA, or protein levels were found, indicating more abundant demand for DHP receptor after increased muscle activity.ResumenPara evaluar los cambios inducidos en la expresión de los receptores de dihidropiridina (DHPR) y rianodina (RyR) por el entrenamiento con ejercicio de baja intensidad, se determinan los niveles de mRNA y de proteína mediante el análisis de RT-PCR cuantitativa e inmunoblot de los músculos gastrocnemius (GAS) y rectus femoris (RF) de ratón sometido a un programa de ejercicio aeróbico durante 15 semanas. El nivel de trabajo muscular fue determinado por los cambios en contenido de cadena pesada de miosina (MHC), expresión de mioglobina (Mb) y tamaño del músculo. La cantidad de mRNA y de proteína de DHPR aumentó significativamente en un 66,8 y 39,5% respectivamente. La expresión de RyR, por otro lado, no se vio incrementada. En RF hubo un aumento significativo del 22,7% en la expresión del mRNA de DHPR, aunque los niveles de proteína permanecieron inalterados. Tampoco se observaron cambios en la expresión de RyR en RF. El entrenamiento dio lugar a un aumento del 1,58% en la cantidad de MHC IIa y disminución del 2,34% en MHC IIb y IId en GAS, con incremento significativo del 8,3% en el contenido de Mb. En RF no se detectaron cambios significativos en el contenido en MHC ni en Mb. Nuestros resultados muestran que se induce un evidente aumento en el nivel de RNAm y de proteína DHPR en GAS mediante un ejercicio de relativamente baja intensidad. Sorprendentemente, en contraste con la expresión de DHPR, no se encontraron cambios en los niveles de mRNA ni de proteína de RyR, indicando mayor demanda de DHPR al incrementar la actividad muscular.

Long-term postinfarction melatonin administration alters the expression of DHPR, RyR2, SERCA2, and MT2 and elevates the ANP level in the rat left ventricle

Abstract:  We investigated the effect of 2 wk continuous postinfarction subcutaneous melatonin supply on the expression of the rat left ventricular (LV) dihydropyridine receptor (DHPR), ryanodine receptor (RyR2), and sarco‐endoplasmic reticulum Ca2+‐ATPase2 (SERCA2), as they are fundamental proteins in cardiac contractility. The levels of plasma and LV atrial (ANP) and brain natriuretic peptide and melatonin were also measured, as was the expression of LV MT1 and MT2 receptors and pineal arylalkylamine N‐acetyltransferase. Myocardial infarction (MI) was induced by ligation of the left anterior descending coronary artery and vehicle or melatonin (4.5 mg/kg per day) was administered by subcutaneous osmotic pumps. Echocardiography, real‐time quantitative reverse transcription‐polymerase chain reaction, and western blotting were used to analyze the samples. Echocardiography revealed that MI induced serious systolic LV dysfunction. The expression of DHPR, RyR2, and SERCA2 mRNAs was significantly lower in the LVs of melatonin‐treated MI rats compared with vehicle‐treated rats (P < 0.01 for DHPR and P < 0.05 for RyR2 and SERCA2). Melatonin also elevated the amount of LV MT2 receptors to 1.9‐fold (P < 0.05) and the concentration of LV ANP to over fivefold (P < 0.05) compared with vehicle rats after MI. Therefore, the results suggest that melatonin may influence the cardiac contractility after MI by regulating the expression of DHPR, RyR2, and SERCA2, and melatonin receptors, particularly MT2s, might contribute to the postinfarction cardioprotective actions of melatonin. Furthermore, the finding of the relationship between melatonin and ANP suggests a novel mechanism for melatonin in protecting the heart after MI.