The effects of aging on carbonic anhydrase concentrations in rat liver and skeletal muscle.

The isoenzymes carbonic anhydrase II (CAII) and III (CAIII) have been measured by radioimmunoassay in the livers of male and female rats aged from 21 to 800 days. No sexual dimorphism at 21 days was found, but from 50 to 400 days both isoenzymes show sexual differences. From 600 days onwards, these differences are less apparent. CAIII concentrations in two 'fast' fibre muscles and one 'slow' fibre muscle have been determined. There is no sexual dimorphism in muscle, but a wide variation between individuals was observed. Fast muscles show maximal CAIII levels at 800 days, whereas in slow muscle the concentration of the isoenzyme is declining at this time.     

Myofascial force transmission between a single muscle head and adjacent tissues: length effects of head III of rat EDL.

Force transmission from muscle fibers via the connective tissue network (i.e., myofascial force transmission) is an important determinant of muscle function. This study investigates the role of myofascial pathways for force transmission from multitendoned extensor digitorum longus (EDL) muscle within an intact anterior crural compartment. Effects of length changes exclusively of head III of rat EDL muscle (EDL III) on myofascial force transmission were assessed. EDL III was lengthened at the distal tendon. For different lengths of EDL III, isometric forces were measured at the distal tendon of EDL III, as well as at the proximal tendon of whole EDL and at the distal tendons of tibialis anterior and extensor hallucis longus (TA+EHL) muscles. Lengthening of EDL III caused high changes in force exerted at the distal tendon of EDL III (from 0 to 1.03 +/- 0.07 N). In contrast, only minor changes were found in force exerted at the proximal EDL tendon (from 2.37 +/- 0.09 to 2.53 +/- 0.10 N). Increasing the length of EDL III decreased TA+EHL force significantly (by 7%, i.e., from 5.62 +/- 0.27 to 5.22 +/- 0.32 N). These results show that force is transmitted between EDL III and adjacent tissues via myofascial pathways. Optimal force exerted at the distal tendon of EDL III (1.03 +/- 0.07 N) was more than twice the force expected on the basis of the physiological cross-sectional area of EDL III muscle fibers (0.42 N). Therefore, a substantial fraction of this force must originate from sources other than EDL III. It is concluded that myofascial pathways play an important role in force transmission from multitendoned muscles.     

Alterations in intrinsic mitochondrial function with aging are fiber type-specific and do not explain differential atrophy between muscles

To determine whether mitochondrial dysfunction is causally related to muscle atrophy with aging, we examined respiratory capacity, H(2) O(2) emission, and function of the mitochondrial permeability transition pore (mPTP) in permeabilized myofibers prepared from four rat muscles that span a range of fiber type and degree of age-related atrophy. Muscle atrophy with aging was greatest in fast-twitch gastrocnemius (Gas) muscle (-38%), intermediate in both the fast-twitch extensor digitorum longus (EDL) and slow-twitch soleus (Sol) muscles (-21%), and non-existent in adductor longus (AL) muscle (+47%). In contrast, indices of mitochondrial dysfunction did not correspond to this differential degree of atrophy. Specifically, despite higher protein expression for oxidative phosphorylation (oxphos) system in fast Gas and EDL, state III respiratory capacity per myofiber wet weight was unchanged with aging, whereas the slow Sol showed proportional decreases in oxphos protein, citrate synthase activity, and state III respiration. Free radical leak (H(2) O(2) emission per O(2) flux) under state III respiration was higher with aging in the fast Gas, whereas state II free radical leak was higher in the slow AL. Only the fast muscles had impaired mPTP function with aging, with lower mitochondrial calcium retention capacity in EDL and shorter time to mPTP opening in Gas and EDL. Collectively, our results underscore that the age-related changes in muscle mitochondrial function depend largely upon fiber type and are unrelated to the severity of muscle atrophy, suggesting that intrinsic changes in mitochondrial function are unlikely to be causally involved in aging muscle atrophy.     

The effect of a unilateral muscle transplantation on the muscle fiber type and the MyHC isoform content in unoperated hind limb slow and fast muscles of the inbred Lewis rats

To reveal the effect of foreign innervation and altered thyroid status on fiber type composition and the myosin heavy chain (MyHC) isoform expression in the rat slow soleus (SOL) and fast extensor digitorum longus (EDL) muscles, a method of heterochronous isotransplantation was developed. In this experimental procedure, the SOL or EDL muscles of young inbred Lewis rats are grafted either into the host EDL or SOL muscles of adult rats of the same strain with normal or experimentally altered thyroid status. To estimate the extent of fiber type transitions in the transplanted muscles, the SOL and EDL muscle from the unoperated leg and unoperated muscles from the operated leg could be legitimately used as controls, but only when the experimental procedure itself does not affect these muscles. To verify this assumption, we have compared the fiber type composition and the MyHC isoform content of unoperated contralateral SOL and EDL muscles and ipsilateral unoperated SOL muscle of experimental rats after unilateral isotransplantation into the host EDL muscle with corresponding muscles of the naive rats of the same age and strain. We provide compelling evidence that the unilateral heterochronous isotransplantation has no significant effect on the fiber type composition and the MyHC isoform content of unoperated muscles of experimental animals. Hence, these muscles can be used as controls in our grafting experiments.     

Contractile properties of EDL and soleus muscles of myostatin-deficient mice.

Myostatin is a negative regulator of muscle mass. The impact of myostatin deficiency on the contractile properties of healthy muscles has not been determined. We hypothesized that myostatin deficiency would increase the maximum tetanic force (P(o)), but decrease the specific P(o) (sP(o)) of muscles and increase the susceptibility to contraction-induced injury. The in vitro contractile properties of extensor digitorum longus (EDL) and soleus muscles from wild-type (MSTN(+/+)), heterozygous-null (MSTN(+/-)), and homozygous-null (MSTN(-/-)) adult male mice were determined. For EDL muscles, the P(o) of both MSTN(+/-) and MSTN(-/-) mice were greater than the P(o) of MSTN(+/+) mice. For soleus muscles, the P(o) of MSTN(-/-) mice was greater than that of MSTN(+/+) mice. The sP(o) of EDL muscles of MSTN(-/-) mice was less than that of MSTN(+/+) mice. For soleus muscles, however, no difference in sP(o) was observed. Following two lengthening contractions, EDL muscles from MSTN(-/-) mice had a greater force deficit than that of MSTN(+/+) or MSTN(+/-) mice, whereas no differences were observed for the force deficits of soleus muscles. Myostatin-deficient EDL muscles had less hydroxyproline, and myostatin directly increased type I collagen mRNA expression and protein content. The difference in the response of EDL and soleus muscles to myostatin may arise from differences in the levels of a myostatin receptor, activin type IIB. Compared with the soleus, the amount of activin type IIB receptor was approximately twofold greater in EDL muscles. The results support a significant role for myostatin not only in the mass of muscles but also in the contractility and the composition of the extracellular matrix of muscles.     

Fiber-type-specific alphaB-crystallin distribution and its shifts with T(3) and PTU treatments in rat hindlimb muscles.

Changes in alphaB-crystallin content in adult rat soleus and extensor digitorum longus (EDL) were examined after 8 wk of 3,5, 3'-triiodothyronine (T(3)) and propylthiouracil (PTU) treatments. Cellular distributions of alphaB-crystallin expression related to fiber type, and distribution shifts with these treatments were also examined in detail from the gray level of reactivity to specific anti-alphaB-crystallin antibody. alphaB-crystallin content in both soleus and EDL muscles was significantly decreased after T(3), and that in EDL was significantly increased over twofold after PTU treatment. In both control soleus and EDL muscles, the gray level of type I fibers was higher than that of type II fibers. alphaB-crystallin expression among type II subtypes was muscle specific; the order was type I > IIa > IIx > IIb in control EDL muscle and type IIx > or = IIa in soleus muscle. The relation was basically unchanged in both muscles after T(3) treatment and was, in particular, well maintained in EDL muscle. Under hypothyroidism conditions with PTU, the mean alphaB-crystallin levels of type IIa and IIx fibers were significantly lower than levels under control conditions. Thus the relation between fiber type and the expression manner of stress protein alphaB-crystallin is muscle specific and also is well regulated under thyroid hormone, especially in fast EDL muscle.     

Chronic stimulation-induced effects point to a coordinated expression of carbonic anhydrase III and slow myosin heavy chain in skeletal muscle

Chronic low-frequency stimulation of rat fast-twitch muscle induces 3.7-fold elevations in cytochrome c oxidase activity, but remains without effect on carbonic anhydrase III (CAIII) mRNA and protein. This is in contrast with the situation in the rabbit where chronic stimulation elicits more than 10-fold elevations in CAIII activity and mRNA content which coincide with an enhanced expression of the slow myosin heavy chain (HCI). Since chronic stimulation of rat muscle does not enhance the expression of HCI, we conclude that CAIII is expressed in parallel with HCI and, therefore, is present only in type I and C fibers.     

Activity dependent characteristics of fast and slow muscle: biochemical and histochemical considerations

The effects of denervation and hindlimb suspension induced disuse on concentrations of ATP, phosphocreatine (PC), and fiber type profile were investigated in slow twitch soleus and fast twitch extensor digitorum longus (EDL) muscles. The results show that the soleus and EDL muscles differ in their dependency on loadbearing as a stimulus for maintaining normal energy metabolism and the biochemical and morphological characteristics of muscle fibers. As determined by R-P methodology, suspension reduced ATP and PC concentrations of the soleus to 26% and 56%, respectively, while, in EDL only, PC is reduced to 71% of control with no change in ATP. Both muscles, however, show identical losses in ATP and PC following denervation. The energy charge, an indicator of Pi availability in muscle was reduced significantly in both denervated muscles to 82% and 85% in soleus and EDL, respectively. No significant reduction of the energy charge was seen in the muscles from suspended rats. Thus, in parallel with the indirect regulation through muscle loadbearing, the nerve can effectively modulate the levels of high-energy phosphates more directly by some regulatory mechanisms independent of muscle type. Denervation and suspension disuse increased the proportion of type 2 fibers in the soleus with a concomitant decrease in type 1 fibers and a relative rise in the number of very small diameter fibers. The EDL showed only variation in fiber size.     

Hypophysectomy abolishes sexual dimorphism of liver carbonic anhydrase III

Hypophysectomy was found to have no effect on the concentration of carbonic anhydrase III (CAIII) in male rat liver, whereas in the female, CAIII was elevated 10-fold, to male levels.     

Androgen-linked control of rat liver carbonic anhydrase III

The concentration of carbonic anhydrase III (CAIII) in male rat liver was found to be 30 times greater than that in the female. Castration of male rats led to marked reduction in liver CAIII concentrations which could be partially restored to control levels by testosterone replacement. Marked developmental and senescence changes in liver CAIII were also observed in male rats.     

Carbonic anhydrase III regulates peroxisome proliferator-activated receptor-γ2

Carbonic anhydrase III (CAIII) is an isoenzyme of the CA family. Because of its low specific anhydrase activity, physiological functions in addition to hydrating CO(2) have been proposed. CAIII expression is highly induced in adipogenesis and CAIII is the most abundant protein in adipose tissues. The function of CAIII in both preadipocytes and adipocytes is however unknown. In the present study we demonstrate that adipogenesis is greatly increased in mouse embryonic fibroblasts (MEFs) from CAIII knockout (KO) mice, as demonstrated by a greater than 10-fold increase in the induction of fatty acid-binding protein-4 (FABP4) and increased triglyceride formation in CAIII(-/-) MEFs compared with CAIII(+/+) cells. To address the underlying mechanism, we investigated the expression of the two adipogenic key regulators, peroxisome proliferator-activated receptor-γ2 (PPARγ2) and CCAAT/enhancer binding protein-α. We found a considerable (approximately 1000-fold) increase in the PPARγ2 expression in the CAIII(-/-) MEFs. Furthermore, RNAi-mediated knockdown of endogenous CAIII in NIH 3T3-L1 preadipocytes resulted in a significant increase in the induction of PPARγ2 and FABP4. When both CAIII and PPARγ2 were knocked down, FABP4 was not induced. We conclude that down-regulation of CAIII in preadipocytes enhances adipogenesis and that CAIII is a regulator of adipogenic differentiation which acts at the level of PPARγ2 gene expression.     

Identification of the zinc-binding protein specifically present in male rat liver as carbonic anhydrase III

A zinc (Zn)-binding protein that is present specifically in the livers of male adult rats was detected by HPLC with in-line detection by mass spectrometry (ICP MS). The Zn-binding protein was purified on Sephadex G-75 and G3000SW HPLC columns. and was identified as carbonic anhydrase III (CAIII) based on the amino acid sequence of a peptide obtained on lysyl endopeptidase digestion. CAIII is expressed as one of the major Zn-binding proteins in the livers of male rats in an age-dependent manner, a comparable amount of Zn to that of copper, Zn-superoxide dismutase (Cu,Zn-SOD) being bound to CAIII at 8 weeks of age. Castration at 4 or 8 weeks of age was shown to reduce Zn bound to CAIII to 47.5% of the sham-operated control level, suggesting that the sex-dependent expression of CAIII is partly regulated by a sex hormone, androgen. The concentration of CAIII in the livers of Long-Evans rats with a cinnamon-like coat color (LEC rats), an animal model of Wilson disease, was also estimated as Zn bound to CAIII and shown to be lower than that in Wistar rats before the onset of hepatitis. The concentration of CAIII was decreased specifically by repeated injections of cupric ions without the Cu,Zn-SOD concentration being affected.     

Activity of some enzymes of energy metabolism during denervation and reflex atrophy in rat slow and fast muscles

The loss of muscle weight in the soleus (SOL) and extensor digitorum longus (EDL) muscles was compared after denervation and in the course of reflex muscle atrophy induced by unilateral fracture of metatarsal bones of the paw and local injection of 0.02 ml turpentine oil subcutaneously. This so-called reflex atrophy is significantly greater after 3 days than that after denervation. Seven days after the nociceptive stimulus, reflex and denervation atrophy are grossly similar in both muscles. This also applies in case that the nociceptive stimulus had been repeated on the third day. The EDL:SOL enzyme activities of energy supply metabolism reflect the differences between a glycolytic-aerobic (EDL) and predominantly aerobic type (SOL) of muscle. No consistent changes were found in either type of atrophy after 3 days. In 7 days' denervation, the activity of hydroxyacetyl-CoA-dehydrogenase (HOADH) and citrate synthase (CS) was decreased in the SOL, while glycerolphosphate:NAD dehydrogenase (GPDH) was enhanced. In the EDL, the activity of triosephosphate dehydrogenase (TPDH), GPDH, malate dehydrogenase (MDH), CS and HOADH was decreased. Acid phosphatase (AcP) was greatly increased in both muscles. Seven days after application of the nociceptive stimulus, all enzyme activities were altered in a grossly analogous manner as after denervation.     

Fiber number and type composition in extensor digitorum longus, soleus, and diaphragm muscles with aging in Fisher 344 rats

Histochemical (M-ATPase) fiber typing was done on extensor digitorum longus, (EDL), soleus (SOL), and diaphragm (DIA) muscles of barrier-reared Fisher 344 rats obtained at four different ages (3, 9, 28, and 30 months) from the colonies of the National Institute of Aging. In the EDL there are no differences in the percent of type I fibers among the four age groups. The percent of type IIa and IIb fibers also showed no difference between the 3 and 30 month age groups. There was no apparent trend for an increase or decrease in the percent of type IIa or IIb fibers between the four age groups. In both the SOL and DIA muscles the percent of type I fibers was greater in the aged than in the young groups. The percent of type IIa fibers was lower in the 30 month group than in the younger groups for both muscles. The percent of type IIb (DIA) and IIc (SOL) fibers did not change between groups. Total fiber number per cross section of muscle showed no change in the EDL over this age range or in the SOL after 9 months of age. These findings bring into question published results that imply that decreasing fiber number and preferential loss of type II (a and b) fibers are typical aging phenomena.     

Fibre type specificity of haem oxygenase-1 induction in rat skeletal muscle.

The expression of the inducible haem oxygenase (HO-1) gene was examined in different skeletal muscles. Rats were treated with haemin and a time course of HO-1 mRNA expression was determined in soleus and extensor digitorum longus (EDL) muscles. Fibre type composition and tissue myoglobin content were also measured. We found that HO-1 mRNA expression markedly increased in soleus (type I fibres) muscle but was only slightly affected in EDL (type II fibres). HO-1 expression directly correlated with both percentage of red fibres and tissue myoglobin. These data demonstrate that HO-1 gene expression follows a fibre type-specific pattern which might indicate an important role for this protein in the maintenance of skeletal muscle function.     

Comparison of red and white muscle wet weight changed by age, denervation and morbid states

[Na]i, [K]i and wet weight of the extensor digitrum longus (EDL) and soleus (SOL) muscles of 9- and 52-week-old rats were measured for 7 days after sectioning of the sciatic nerve. The changes in wet weight of the EDL and SOL muscles of rats over 52 weeks and those of morbid state rats were also measured. There was no significant difference in wet weights between the EDL and SOL muscles in infant rats, but the EDL muscle became much heavier than the SOL muscle with aging. The decrease in rate of growth of wet weight of the EDL and SOL muscles caused by denervation, was greater in young rats than in mature rats. In addition, the rate of decrease was greater in the SOL muscles than in the EDL muscles in both young and mature rats. The [Na]i increased while [K]i was decreased by denervation, and the net Na+ increase and the net K+ loss were greater in young rats than in mature rats. The changing rate was more remarkable in the EDL muscles than in the SOL muscles throughout the aging process. During DOCA treatment over 4 weeks, the decrease of muscle wet weight was greater in the EDL muscles. The mechanisms which serve to maintain normal muscle wet weight in the SOL muscle after denervation or treatment with DOCA, were discussed.     

Fate of 3H-thymidine labelled myogenic cells in regeneration of muscle isografts

Summary Intact and denervated extensor digitorum longus (EDL) muscles of 20-day-old inbred Lewis-Wistar rats were labelled with ³H-thymidine. Ninety minutes after the injection of the isotope 4.0% of the nuclei were labelled in the intact (i.e. innervated) and 9.6% in the muscles, denervated 3 days before administration of the isotope. The labelled EDL muscles were grafted into the bed of the previously removed EDL muscles of inbred animals and these isografts were studied 30 days later.In the EDL muscles, regenerated from innervated isografts only occasionally labelled endothelial cells were found whereas in the muscles regenerated from denervated isografts also parenchymal muscle nuclei were regularly labelled. The incidence of labelled nuclei in the regenerated EDL muscles was, however, about 20 times lower than in the donor EDL muscles. The present experiments provide a direct proof of utilization of donor satellite cell nuclei for regeneration in grafted muscle tissue. With respect to the low incidence of labelled nuclei in regenerated EDL muscles, other sources of cells apparently also contribute to the regeneration process.