Investigation of changes in skeletal muscle α-actin expression in normal and pathological human and mouse hearts

We have developed a quantitative antibody-based assay to measure the content of skeletal muscle α-actin relative to cardiac α-actin. We found 21 ± 2% skeletal muscle α-actin content in normal heart muscle of adult man and mouse. In end stage failing heart 53 ± 5% of striated actin was skeletal muscle α-actin and in samples of inter-ventricular septum from patients with hypertrophic obstructive cardiomyopathy (HOCM) skeletal muscle α-actin was 72 ± 2% of sarcomeric actin. Thin filaments containing actin isolated from normal and HOCM heart muscle were functionally indistinguishable when studied by quantitative in vitro motility assay. We also found elevated skeletal muscle α-actin (60 ± 7%) in a mouse model of dilated cardiomyopathy.     

Differential expression of α-enolase in the normal and pathological cardiac growth

Objectives

It was found that α-enolase was dramatically up-regulated in the hypertrophic hearts of SHR in our previous study. The purposes of this study were to examine the expression pattern of α-enolase in pre- and postnatal myocardium of spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats, and to explore the relationship between the overexpression of α-enolase and left ventricular hypertrophy.

Methods

HE staining was used for the measurement of cardiac hypertrophy. Immunohistochemical technique was used to evaluate the location of α-enolase. The expressions of α-enolase in the left cardiac ventricles at different development times were examined by Real-time RT-PCR and Western blot.

Results

Cardiac hypertrophy was found in SHR rats at 4 weeks of age and remained up to 24 weeks of age. The signals of α-enolase protein were strong and existed extensively in hypertrophic myocardium in SHR, while in the normal myocardium of WKY, the signals were scarcely found and weak. The levels of α-enolase mRNA and protein in SHR and WKY hearts during fetal stage and newborn stage were similar, while from 4 weeks of age to 24 weeks of age, accompanied by the cardiac hypertrophy, the levels of α-enolase mRNA and protein in left ventricle of SHR were significantly higher than that in WKY.

Conclusions

The expressions of α-enolase in the left ventricle of the rats during normal and pathological cardiac development were different. This phenomenon provides the potential clues to understanding pathophysiological mechanisms in cardiac hypertrophy of SHR.     

Differential response of the membrane systems involved in excitation–contraction coupling to early and later postnatal denervation in rat skeletal muscle

We compared the morphological features of the membrane systems involved in excitation–contraction (E–C) coupling during early postnatal development stages in rat skeletal muscles (tibialis anterior) denervated either at birth or 7 days after birth. Four obvious structural changes are observed in the arrangement of the transverse (T) tubule network and the disposition of triads following early postnatal denervation: (1) an increase in the longitudinal segments of the T tubule network, (2) changes in the direction and disposition of triads, (3) the appearance of caveolae clusters, (4) the appearance of pentads and heptads (i.e. a close apposition of two or three T tubule elements with three or four elements of terminal cisternae of sarcoplasmic reticulum). The increased presence of longitudinal T tubules parallels the loss of cross striations, and this in turn is due to misalignment of the myofibrils. The clusters of caveolae appear almost exclusively in muscle fibres denervated at birth, and pentads and heptads are more frequently observed in muscles denervated at 7 days. The differential growth of muscle fibres in response to denervation leads to the formation of abnormal membrane systems involved in the E–C coupling with very unique morphological features, which differ from the case of denervation in adult muscle fibres.     

Regulation of ubiquitin–proteasome system, caspase enzyme activities, and extracellular proteinases in rat soleus muscle in response to unloading

In the present study, we determined the impact of 5 and 10 days of muscle deconditioning induced by hindlimb suspension (HS) on the ubiquitin–proteasome system of protein degradation and caspase enzyme activities in rat soleus muscles. A second goal was to determine whether activities of matrix metalloproteinase-2/9 (MMP-2/9) and urokinase-type/tissue-type plasminogen activator (PAs) were responsive to HS. As expected, HS led to a pronounced atrophy of soleus muscle. Level of ubiquitinated proteins, chymotrypsin-like activity of 20S proteasome, and Bcl-2-associated gene product-1 protein level were all transitory increased in response to 5 days of HS. These changes may thus potentially account for the decrease in muscle mass observed in response to 5 days of HS. Caspase-3 activity was significantly increased throughout the experimental period, whereas activities of caspase-6, another effector caspase, and caspase-9, the mitochondrial-dependent activator of both caspase-3 and -6, were only increased in response to 10 days of HS. This suggests that caspase-3 may be regulated through mitochondrial-independent and mitochondrial-dependent mechanisms in response to HS. Finally, MMP-2/9 activities remained unchanged, whereas PAs activities were increased after 5 days of HS. Overall, these data suggest that time-dependent regulation of intracellular and extracellular proteinases are important in setting the new phenotype of rat soleus muscle in response to HS.     

Evidence of the co-activation of α-motoneurones and static γ-motoneurones of the sartorius medialis muscle during locomotion in the thalamic cat

Summary The activity in and efferent axon populations and in group I and group II afferent fibre populations innervating a flexor muscle, the sartorius medialis, was observed during spontaneous locomotor movements in the thalamic cat. Multi-unit discharges of each kind of fibre were obtained by electronic sorting of the action potentials from the overall activity of a thin, intact branch of the sartorius medialis nerve. The following results were obtained: (1) The -motoneurones have a phasic behaviour characterized by a single discharge period during the hip flexion (swing phase of the step-cycle). (2) The -motoneurones are co-activated with the homonymous -motoneurones. (3) Between rhythmic and discharges, i.e. during the hip extension (stance phase of the step cycle), both - and -motoneurones were normally silent. However, in 5 out of 17 experiments, a few units of the population fired at very low frequency. (4) Two observations indicate that the -motoneurones that are co-activated with the -motoneurones by central locomotor commands are predominantly of the static type. In actual locomotion, the rhythmic fusimotor discharges over-compensate the depressor effect on the firing rate of the group II afferents of the unloading of muscle spindles by the active shortening of the parent muscle. In fictive locomotion, when the transmission of the excitation is blocked by selective curarization in alpha skeleto-motor junctions alone, the rhythmic fusimotor discharges elicit in-phase modulations not only of the group I but also of the group II fibres. The group II afferent population consists almost entirely of fibres arising from spindle secondary endings which are located primarily on intrafusal muscle fibres whose contraction is exclusively controlled by static fusimotor motoneurones. In the two experimental circumstances, the analysis of the group I fibre discharge does not allow to decide whether dynamic motoneurones are firing or silent during rhythmic discharge. (5) The group I and group II afferent discharges during the step-cycle showed two frequency peaks, one static-fusimotor dependent while the contracting muscle shortened during the hip flexion (swing) phase, the other length-change dependent while the relaxed muscle was rapidly stretched during the first part of the hip extension (stance) phase. Then, during the second part of hip extension when the muscle was slowly stretched in the absence of fusimotor drive, the firing rate of the spindle afférents decreased to a low level. The spindle sensory endings during the extension phase showed low dynamic and static responsiveness like deefferented spindles. (6) The results obtained in sartorius medialis (flexor) muscle are discussed in comparison with the results previously obtained in gastrocnemii (extensor) muscles (Bessou et al. 1986). The consequences of the predominant activation of the static or dynamic fusimotor system in functionally different muscles are considered with respect to the proprioceptive or motor role of musclespindles during muscle contraction.     

Contribution to the epidemiology of postnatal depression in Germany – implications for the utilization of treatment

Summary Using a longitudinal screening model, 772 mothers were screened for postnatal depression after delivery in Stuttgart (Germany). This model contained the Edinburgh Postnatal Depression Scale (EPDS) and the Hamilton Depression Scale (HAMD). The first screening was 6–8 weeks after delivery with the EPDS. Mothers with high scores in the first screening had a second screening 9–12 weeks after delivery with the EPDS at least three weeks after the first. Mothers with high scores in both screenings were investigated with the Hamilton Depression Scale (HAMD).Classification was performed with the DSM-IV. After observation until the third month after delivery, 3.6% (N = 28) of the 772 mothers were diagnosed with postnatal depression. Various methods of therapy were offered to those mothers. 18% (N = 5) accepted one or more of these methods of treatment. The rest of the mothers with postnatal depression refused – mostly for attitudinal or practical reasons. 13.4% of the mothers showed high scores in the first screening but not in the second. For those mothers a longitudinal observation is currently being performed to distinguish between a depressive episode and a depression with oscillating symptoms.     

Significance of peripheral afferent input to the α-motoneurone pool for enhancement of tremor during an isometric fatiguing contraction

The objective of this study was to investigate the contribution of peripheral afferent input to the enhancement of isometric tremor during a sustained submaximal isometric contraction. It was hypothesised that during muscle fatigue, when excitatory drive is high, peripheral afferent input may augment oscillations in the stretch reflex arc and result in bursting motor-unit activity and increased tremor. Nine healthy subjects maintained isometric plantar flexions at 30% of their maximum voluntary contraction until the limit of endurance, under three test conditions. Two paradigms were used to reduce afferent input to the triceps surae α-motoneurone pool: (1) continued vibration of the Achilles tendon, and (2) ischaemic partial block of the tibial nerve. These were compared to a control experiment, in which there was no intervention. By recording H-reflexes from the gastrocnemius and soleus muscles, it was possible to assess the effectiveness of reducing the afferent input. When H-reflex suppression had stabilised, the fatiguing contraction was commenced and tremor was computed from the continuously recorded torque signal. Superimposed maximum twitches were elicited as indirect measures of excitatory drive. The increase in tremor root mean square throughout the fatiguing contraction was significantly less for both the vibration and ischaemic conditions. Furthermore, tremor mean power frequency decreased significantly with endurance time in the control experiment, while no significant change was seen in the other two experimental conditions. It is concluded that the enhancement of isometric tremor seen during a fatiguing submaximal isometric contraction is facilitated by peripheral afferent input to the α-motoneurone pool. Accepted: 9 February 2000     

The effects of running exercise on oxidative capacity and PGC-1α mRNA levels in the soleus muscle of rats with metabolic syndrome

Skeletal muscles in animals with metabolic syndrome exhibit reduced oxidative capacity. We investigated the effects of running exercise on fiber characteristics, oxidative capacity, and mRNA levels in the soleus muscles of rats with metabolic syndrome [SHR/NDmcr-cp (cp/cp); CP]. We divided 5-week-old CP rats into non-exercise (CP) and exercise (CP-Ex) groups. Wistar-Kyoto rats (WKY) were used as the control group. CP-Ex rats were permitted voluntary exercise on running wheels for 10 weeks. Triglyceride levels were higher and adiponectin levels lower in the CP and CP-Ex groups than in the WKY group. However, triglyceride levels were lower and adiponectin levels higher in the CP-Ex group than in the CP group. The soleus muscles in CP-Ex rats contained only high-oxidative type I fibers, whereas those in WKY and CP rats contained type I, IIA, and IIC fibers. Muscle succinate dehydrogenase (SDH) activity was higher in the CP-Ex group than in the CP group; there was no difference in SDH activity between the WKY and CP-Ex groups. Muscle proliferator-activated receptor γ coactivator-1α (PGC-1α) mRNA levels were higher in the CP-Ex group than in the CP group; there was no difference in PGC-1α mRNA levels between the WKY and CP-Ex groups. In CP-Ex rats, longer running distance was associated with increased muscle SDH activity and PGC-1α mRNA levels. We concluded that running exercise restored decreased muscle oxidative capacity and PGC-1α mRNA levels and improved hypertriglyceridemia in rats with metabolic syndrome.     

Place of genotyping in addition to the phenotype and the assay of serum α-1 antitrypsin

The diagnosis of deficiency of alpha-1 antitrypsin (A1AT) is based on isoelectric focusing of serum proteins and the extent of serum. However, the focusing is technically difficult and a greatly reduced concentration in abnormal A1AT tapeless does not differentiate an unstable variant of a variant called 'null' (that is to say without any phenotypic expression) to 'heterozygous' state. In this study, we compared the results of the assay, the phenotype and genotype of A1AT in 50 patients. Normal A1AT alleles (Pi*M1 to Pi*M4) or loss of the most common (Pi*S and Pi*Z) were clearly identified in phenotyping. However, genotyping was necessary to characterize: (i) certain alleles rarer A1AT (S-Munich, X-Christchurch); (ii) a null allele and; (iii) two new alleles A1AT not yet described in the literature. In conclusion, although the A1AT genotyping is generally not necessary, it is necessary to resolve complex cases and to obtain witnesses validated for isoelectric focusing.     

Value of α-smooth muscle actin and glial fibrillary acidic protein in predicting early hepatic fibrosis in chronic hepatitis C virus infection

Introduction

α-Smooth muscle actin (α-SMA)-positive hepatic stellate cells (HSCs) are pericytes responsible for fibrosis in chronic liver injury. The glial fibrillary acidic protein (GFAP), commonly expressed by astrocytes in the central nervous system, is expressed in vivo in the liver in a subpopulation of quiescent stellate cells. The reports concerning GFAP expression in human liver are still conflicting. The aim of the study is investigation the utility of GFAP compared to α-SMA as an indicator of early activated HSCs, in predicting fibrosis in chronic hepatitis C (CHC) patients.

Material and methods

With immunohistochemistry and a semi-quantitative scoring system, the expressions of α-SMA and GFAP on HSCs in liver biopsies from patients with pure CHC (n = 34), hepatitis C virus-induced cirrhosis (n = 24), mixed CHC/schistosomiasis (n = 11) and normal controls (n = 10) were analysed.

Results

The immunoreactivity of α-SMA and GFAP in perisinusoidal, periportal and pericentral areas was assessed. α-Smooth muscle actin and GFAP-positive HSCs were significantly increased in all diseased groups compared with normal controls. In pure CHC with or without cirrhosis, perisinusoidal α-SMA-positive HSCs were predominant in relation to GFAP-positive cells. On the other hand, GFAP-positive cells were predominant in the group of schistosomiasis as compared with the other diseased groups. It was noticed that expression of GFAP on perisinusoidal HSCs in CHC patients sequentially decreased with the progression of fibrosis.

Conclusions

Glial fibrillary acidic protein could represent a more useful marker than α-SMA of early activation of HSCs in CHC patients and seems to be an early indicator of hepatic fibrogenesis.     

Alteration in α- and β- adrenoceptor profile of rabbit-knee-joint blood vessels due to chronic inflammation

Experiments were performed to investigate the nature of α- and β-adrenoceptors in blood vessels supplying the posterior capsule of the chronically inflamed rabbit knee joint, and results were compared to the finding from previous experiments on the normal and acutely inflamed joint to assess any alteration which may occur in the adrenoceptor profile due to the chronic inflammation process. Electrical stimulation of the posterior articular nerve resulted in vasoconstriction that was completely blocked by phentolamine. This constrictor response was almost equally inhibited by prazosin and yohimbine. The dose–response curves to close intraarterial injection of α-adrenoceptor agonists showed a rank-order potency of adrenaline = clonidine = phenylephrine. The adrenaline dose–response curve was shifted to the right by administration of α-antagonists with a rank-order potency of phentolamine = prazosin = yohimbine. At this stage of experiments, there was an equal response of α₁- and α₂- adrenoceptors in blood vessels of the chronically inflamed rabbit knee joint. In another group of animals, the neurally mediated vasodilatation, which appeared after administration of phentolamine, was completely blocked by propranolol and was reduced significantly by ICI118551, but the effect of atenolol was not significant. The dose–response curve to close intraarterial injection of β-adrenoceptor agonists showed a rank-order potency of: isoprenaline > salbutamol > dobutamine. The isoprenaline dose–response curve was shifted to the right by administration of β-antagonists with rank-order potency of propranolol > ICI118551 > atenolol. These experiments showed a greater β₂-adrenoceptor response than β₁-adrenoceptor response in chronically inflamed rabbit-knee-joint blood vessels. Overall, compared to previous experiments on normal joint in which α₂- and β₁-adrenoceptor responses predominated, and in acutely inflamed joint in which an equal α₁/α₂ and β₁/β₂ response was shown, in chronically inflamed joint the sympathetic constriction response was returned toward normal. No more α-adrenoceptor shift had happened, and the shift of β₁ to β₂ response continued.     

Cross-sectional Anatomy and Clinical Meanings of the Extraocular Muscles and Related Structures in Orbit

1IntroductionWith the development of modern medical technolo-gy,such as CT, MRI , which has become indispensablediagnostic tool in ophthalmology,the needs of thin sec-tions ,which compared with CTor MRI are more than ev-er .There are some sections have been made ,buttheyarethick sections ,usually thicker than 5 mm, which can’tdisplaythe structures in orbit well .In our research,con-tinuous slices ,0·5 mmthickness ,were sectionedincoro-nal plane with celloidin embedding process and observ…     

Expression and regulation of αB-crystallin in the kidney in vivo and in vitro

αB-crystallin, a major component of the mammalian eye lens, is a small heat shock protein and molecular chaperone that is also abundant in the mammalian kidney. The present study aimed to characterize more closely the intrarenal expression and regulation of αB-crystallin in vivo and in vitro. In normal rat kidney, the expression of αB-crystallin mRNA and protein were both close to the detection limit in cortex, but increased steeply from the outer to the inner medulla where αB-crystallin constitutes approximately 2% of total tissue protein. Immunohistochemistry disclosed papillary collecting duct cells and thin limbs as the major sites for intrapapillary αB-crystallin immunoreactivity. In rats subjected to sucrose diuresis for 3 days, αB-crystallin mRNA expression was reduced by 27 and 46% in outer and inner medulla, respectively. In agreement with the results obtained in vivo, in Madine–Darby canine kidney cells, αB-crystallin mRNA and protein were induced significantly by elevating the medium osmolality to 500 mosm/kg H₂O by the addition of NaCl and raffinose, and also by urea. The NaCl-induced increase in αB-crystallin expression was concentration-dependently blunted by SP600125, a specific JNK inhibitor. Overexpression of αB-crystallin in 293 cells resulted in increased tolerance to acute osmotic stress. These results indicate that αB-crystallin may be regulated by papillary interstitial tonicity in a JNK-dependent process. Moreover, the high abundance of αB-crystallin in the renal medulla may be important for cell survival in an environment characterized by extreme interstitial solute concentrations as present during antidiuresis.     

Regulation of the L-type calcium channel α-1 subunit by chronic depolarization in the neuron-like PC12 and aortic smooth muscle A7r5 cell lines

The regulation of L-type voltage-dependent Ca²⁺ channels by chronic depolarization was studied in the aortic smooth muscle A7r5 and neuron-type PC12 cell lines, by probing the expression and the functional state of their constitutive -1 subunits. PC12 cells showed, after prolonged exposure to a high-K⁺ depolarizing solution, a 25% reduction of the functional Ca²⁺ channel density which was accompanied by a decrease of the -1 subunit mRNA expression. In A7r5 cells submitted to a similar protocol of depolarization, ⁴⁵Ca²⁺ uptake measurements revealed a fall in the functional activity of L-type Ca²⁺ channels which was not related to a modulation of their mRNA expression, but arose from a long-term voltage-dependent channel inactivation. Accordingly, the lag time and the mechanisms of recovery were different in the two cell types. In PC12 cells, when restoring physiological culture conditions, de novo synthesis of -1 subunits allowed the recovery of the original density of L-type Ca²⁺ channels at the membrane surface. As for the A7r5 cells, we showed that after chronic depolarization, the complete restoration of the resting membrane potential and the related Ca²⁺ channel activity required a 2-day incubation in physiological medium and could probably be related to a normalization of the increased intracellular Ca²⁺ concentration. In contrast, it is noteworthy that, in PC12 cells, the only transient increase of intracellular Ca²⁺ content in the first hours of depolarization could account for the long-term down-regulation of L-type Ca²⁺ channels.     

Testing the plasticity of insulin secretion and β-cell function in vivo: responses to chronic hyperglycaemia in the sheep

Plasticity of insulin secretion is essential to maintain the action of insulin during insulin resistance and to prevent diabetes. Investigation of the plasticity of insulin secretion and its regulation is challenging, and the objective of this study was to develop a novel large-animal-based model. The effect of chronic moderate hyperglycaemia on the plasticity of insulin secretion, β-cell mass and function was determined in sheep. Adolescent sheep (120 days old) were infused with 25% glucose for 16 days to increase blood glucose by 50% (n = 10), and control animals (n = 9) were infused with saline. Glucose- and arginine-stimulated insulin secretion, insulin sensitivity and glucose effectiveness were measured in vivo before and during treatment (days 10-14), and β-cell mass was measured at the end of treatment. Hyperglycaemia increased blood glucose (+53%) and plasma insulin (+403%; each P < 0.003) and did not alter whole-body insulin sensitivity. Hyperglycaemia increased glucose-stimulated insulin secretion (particularly second phase; five-fold) and arginine-stimulated insulin secretion (particularly first phase; four-fold). Hyperglycaemia reduced β-cell mass (～50%, P = 0.038) and increased glucose- and arginine-stimulated insulin secretion relative to β-cell mass five-fold (P = 0.060) and 20-fold (P = 0.007), respectively. Chronic hyperglycaemia therefore induces marked adaptation and upregulation of glucose- and arginine-stimulated insulin secretion by enhancing β-cell function rather than increasing β-cell mass in the sheep, consistent with long-term adaptations seen in humans. This marked plasticity of insulin secretion in response to moderate hyperglycaemia provides a novel model for the investigation of factors affecting its capacity and underlying determinants.     

Glycine- and GABA-immunoreactive nerve terminals apposed to α-motoneurons during postnatal development in kittens: a quantitative study using the disector

The distribution of γ-aminobutyric acid (GABA) and/or glycine-immunoreactive (IR) terminal-like structures apposed to somatic and dendritic membrane of lumbar α-motoneurons in column 2 was examined in 1- and 3-week-old kittens and in the adult cat. This quantitative study was carried out using a postembedding technique on semithin sections and a stereological method, the disector. Analysis of immunoreactive terminals showed that the percentages of GABA-IR and glycine-IR terminals (these populations include terminals containing both GABA and glycine) apposed to somatic and proximal dendritic compartments of α-motoneurons are almost the same in kittens, while in the adult glycinergic innervation becomes predominant. This change results from: (1) the decrease in numbers of GABA-IR terminals contacting the somatic compartment between 3 weeks and adult stage, while the numbers of glycine-IR terminals show no significant changes after birth and the numbers of terminals containing both neurotransmitters (GABA-IR+glycine-IR) present transient changes and (2) the postnatal increase in the dendritic compartment, in numbers of GABA-IR, glycine-IR and GABA-IR+glycine-IR terminals; the increase being larger for glycine-IR terminals. Furthermore, using a postembedding immunogold technique, observations by electron microscopy showed that GABA-IR P boutons apposed to M boutons can already be identified at 1 week after birth. Received: 1 June 1998 / Accepted: 6 May 1999