Proteasome inhibition improves diaphragm function in an animal model for COPD

Diaphragm muscle weakness in patients with chronic obstructive pulmonary disease (COPD) is associated with increased morbidity and mortality. Recent studies indicate that increased contractile protein degradation by the proteasome contributes to diaphragm weakness in patients with COPD. The aim of the present study was to investigate the effect of proteasome inhibition on diaphragm function and contractile protein concentration in an animal model for COPD. Elastase-induced emphysema in hamsters was used as an animal model for COPD; normal hamsters served as controls. Animals were either treated with the proteasome inhibitor Bortezomib (iv) or its vehicle saline. Nine months after induction of emphysema, specific force-generating capacity of diaphragm bundles was measured. Proteolytic activity of the proteasome was assayed spectrofluorometrically. Protein concentrations of proteasome, myosin, and actin were measured by means of Western blotting. Proteasome activity and concentration were significantly higher in the diaphragm of emphysematous hamsters than in normal hamsters. Bortezomib treatment reduced proteasome activity in the diaphragm of emphysematous and normal hamsters. Specific force-generating capacity and myosin concentration of the diaphragm were reduced by ~25% in emphysematous hamsters compared with normal hamsters. Bortezomib treatment of emphysematous hamsters significantly increased diaphragm-specific force-generating capacity and completely restored myosin concentration. Actin concentration was not affected by emphysema, nor by bortezomib treatment. We conclude that treatment with a proteasome inhibitor improves contractile function of the diaphragm in emphysematous hamsters through restoration of myosin concentration. These findings implicate that the proteasome is a potential target of pharmacological intervention on diaphragm weakness in COPD.     

bHLH transcription factor MyoD affects myosin heavy chain expression pattern in a muscle-specific fashion

A strong correlative pattern between MyoD gene expression and myosin heavy chain IIB (MHC IIB) gene expression exists. To test whether this correlative relationship is causative, MHC gene expression in muscles from MyoD(-/-) mice was analyzed. The MHC IIB gene was not detectable in the MyoD(-/-) diaphragm, whereas the MHC IIB protein made up 10.0 +/- 1.7% of the MHC protein pool in the wild-type (WT) mouse diaphragm. Furthermore, the MHC IIA protein was not detectable in the MyoD(-/-) biceps brachii, and the MHC IIB protein was overexpressed in the masseter. To examine whether MyoD is required for the upregulation of the MHC IIB gene within slow muscle after disuse, MyoD(-/-) and WT hindlimb musculature was unweighted. MyoD(-/-) exhibited a diminished response in the upregulation of the MHC IIB mRNA within the soleus muscle as a result of the hindlimb unweighting. Collectively, these data suggest that MyoD plays a role in the MHC profile in a muscle-specific fashion.     

Early changes in muscle fiber size and gene expression in response to spinal cord transection and exercise

Muscles ofspinal cord-transected rats exhibit severe atrophy and a shift toward afaster phenotype. Exercise can partially prevent these changes. Thegoal of this study was to investigate early events involved inregulating the muscle response to spinal transection and passivehindlimb exercise. Adult female Sprague-Dawley rats were anesthetized,and a complete spinal cord transection lesion(T₁₀) was created in all ratsexcept controls. Rats were killed 5 or 10 days after transection orthey were exercised daily on motor-driven bicycles starting at 5 daysafter transection and were killed 0.5, 1, or 5 days after the firstbout of exercise. Structural and biochemical features of soleus andextensor digitorum longus (EDL) muscles were studied. Atrophy wasdecreased in all fiber types of soleus and in type 2a and type 2xfibers of EDL after 5 days of exercise. However, exercise did notappear to affect fiber type that was altered within 5 days of spinalcord transection: fibers expressing myosin heavy chain 2xincreased in soleus and EDL, and extensive coexpression of myosin heavy chain in soleus was apparent. Activation of satellite cells was observed in both muscles of transected rats regardless of exercise status, evidenced by increased accumulation of MyoD and myogenin. Increased expression was transient, except for MyoD, which remained elevated in soleus. MyoD and myogenin were detected both in myofiber and in satellite cell nuclei in both muscles, but in soleus, MyoD waspreferentially expressed in satellite cell nuclei, and in EDL, MyoD wasmore readily detectable in myofiber nuclei, suggesting that MyoD andmyogenin have different functions in different muscles. Exercise didnot affect the level or localization of MyoD and myogenin expression.Similarly, Id-1 expression was transiently increased in soleus and EDLupon spinal cord transection, and no effect of exercise was observed.These results indicate that passive exercise can ameliorate muscleatrophy after spinal cord transection and that satellite cellactivation may play a role in muscle plasticity in response to spinalcord transection and exercise. Finally, the mechanisms underlyingmaintenance of muscle mass are likely distinct from those controllingmyosin heavy chain expression.

     

Altered expression of skeletal muscle myosin isoforms in cancer cachexia

Cachexia is commonly seen in cancer and ischaracterized by severe muscle wasting, but little is known about theeffect of cancer cachexia on expression of contractile protein isoforms such as myosin. Other causes of muscle atrophy shift expression ofmyosin isoforms toward increased fast (type II) isoform expression. Weinjected mice with murine C-26 adenocarcinoma cells, a tumor cell linethat has been shown to cause muscle wasting. Mice were killed 21 daysafter tumor injection, and hindlimb muscles were removed. Myosin heavychain (MHC) and myosin light chain (MLC) content was determined inmuscle homogenates by SDS-PAGE. Body weight was significantly lower intumor-bearing (T) mice. There was a significant decrease in muscle massin all three muscles tested compared with control, with the largestdecrease occurring in the soleus. Although no type IIb MHC was detectedin the soleus samples from control mice, type IIb comprised 19% of thetotal MHC in T soleus. Type I MHC was significantly decreased in T vs. control soleus muscle. MHC isoform content was not significantly different from control in plantaris and gastrocnemius muscles. Thesedata are the first to show a change in myosin isoform expression accompanying muscle atrophy during cancer cachexia.

     

Contractile properties of expiratory abdominal muscles: effect of elastase-induced emphysema

The present study examined the intrinsic contractile properties and endurance of the transverse abdominis and external oblique abdominal expiratory muscles in adult hamsters and compared their performance with the diaphragm. Experiments were performed in vitro on isolated bundles of muscle stimulated electrically. In control animals peak twitch tension was similar in the two muscles. In contrast, the twitch contraction time and one-half relaxation time of the transverse abdominis were significantly greater than that of the external oblique. The isometric tension generated over a range of stimulus frequencies (i.e., the force-frequency relationship) was a greater percent of the maximum value in response to subtetanizing frequencies (10-40 Hz) in the transverse abdominis than in the external oblique. For both abdominal muscles, however, the tension generated over this range of stimulus frequencies was less than that of the diaphragm. The endurance of the transverse abdominis during repeated contractions was significantly greater than that of the external oblique but similar to the diaphragm. The effect of chronic hyperinflation produced by elastase-induced emphysema on the contractile function of the two muscles was assessed in a second group of adult hamsters. In emphysematous animals peak twitch tension, contraction time, and one-half relaxation time of the twitch and force-frequency curves of muscles from emphysematous animals were similar to values obtained in control animals for both the external oblique and transverse abdominis. However, the endurance of both the transverse abdominis and external oblique muscles was greater in emphysematous than control animals.(ABSTRACT TRUNCATED AT 250 WORDS)     

Functional adaptation of diaphragm to chronic hyperinflation in emphysematous hamsters

Costal strips of diaphragmatic muscle obtained from animals with elastase-induced emphysema generate maximum tension at significantly shorter muscle fiber lengths than muscle strips from control animals. The present study examined the consequences of alterations in the length-tension relationship assessed in vitro on the pressure generated by the diaphragm in vivo. Transdiaphragmatic pressure (Pdi) and functional residual capacity (FRC) were measured in 22 emphysematous and 22 control hamsters 4-5 mo after intratracheal injection of pancreatic elastase or saline, respectively. In 12 emphysematous and 12 control hamsters Pdi was also measured during spontaneous contractions against an occluded airway. To allow greater control over muscle excitation, Pdi was measured during bilateral tetanic (50 Hz) electrical stimulation of the phrenic nerves in 10 emphysematous and 10 control hamsters. Mean FRC in the emphysematous hamsters was 183% of the value in control hamsters (P less than 0.01). During spontaneous inspiratory efforts against a closed airway the highest Pdi generated at FRC tended to be greater in control than emphysematous hamsters. When control hamsters were inflated to a lung volume approximating the FRC of emphysematous animals, however, peak Pdi was significantly greater in emphysematous animals (70 +/- 6 and 41 +/- 8 cmH2O; P less than 0.05). With electrophrenic stimulation, the Pdi-lung volume curve was shifted toward higher lung volumes in emphysematous hamsters. Pdi at all absolute lung volumes at and above the FRC of emphysematous hamsters was significantly greater in emphysematous compared with control animals. Moreover, Pdi continued to be generated by emphysematous hamsters at levels of lung volume where Pdi of control subjects was zero.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential structural features in soleus and gastrocnemius of carnitine-treated cancer cachectic rats

Muscle wasting is associated with chronic diseases and cancer. Elucidation of the biological mechanism involved in the process of muscle mass loss and cachexia may help identify therapeutic targets. We hypothesized that l -carnitine treatment may differentially revert muscle fiber atrophy and other structural alterations in slow- and fast-twitch limb muscles of rats bearing the Yoshida ascites hepatoma. In soleus and gastrocnemius of tumor-bearing rats (10⁸ AH-130 Yoshida ascites hepatoma cells inoculated intraperitoneally) with and without treatment with l -carnitine (1 g/kg body weight for 7 days, intragastric), food intake, body and muscle weights, fiber typing and morphometry, morphological features, redox balance, autophagy and proteolytic, and signaling markers were explored. Levels of carnitine palmitoyl transferase were also measured in all the study muscles. l - Carnitine treatment ameliorated the atrophy of both slow- and fast-twitch fibers (gastrocnemius particularly), muscle structural alterations (both muscles), and attenuated oxidative stress, proteolytic and signaling markers (gastrocnemius). Despite that carnitine palmitoyl transferase-1 levels increased in both muscle types in a similar fashion, l -carnitine ameliorated muscle atrophy and proteolysis in a muscle-specific manner in cancer-induced cachexia. These data reveal the need to study muscles of different fiber type composition and function to better understand whereby l -carnitine exerts its beneficial effects on the myofibers in muscle wasting processes. These findings also have potential clinical implications, since combinations of various exercise and muscle training modalities with l -carnitine should be specifically targeted for the muscle groups to be trained.     

Effect of thoracoabdominal breathing patterns on inspiratory effort sensation

The present study examined the effects of elastase-induced emphysema on the structure of the external oblique and transverse abdominis muscles and a non-respiratory muscle, the extensor digitorum longus. Muscle structure was assessed from the cross-sectional area (CSA) and percent of individual fiber types in histochemically stained sections and from the number of sarcomeres arranged in series along the length of individual fibers. Data were obtained in eight hamsters with emphysema and nine saline-injected controls. In the normal (control) animals the external oblique was thicker but contained fewer sarcomeres than the transverse abdominis. Fiber size was similar in the two muscles. In the transverse abdominis the percents of fast-glycolytic and fast-oxidative fibers were greater and smaller, respectively, than in the external oblique. Lung volume of emphysematous hamsters was 168% of control values (P less than 0.001). In emphysematous compared with control animals, the CSA of fast-twitch fibers in the external oblique and transverse abdominis was significantly reduced. Fiber length and sarcomere number were significantly decreased in the transverse abdominis but not in the external oblique in emphysematous hamsters. In contrast, fiber size and composition of the extensor digitorum longus was similar in emphysematous and control animals. These data indicate that cellular responses of the ventilatory muscles to chronic hyperinflation and altered thoracic geometry induced by emphysema are not present in limb skeletal muscle. We speculate that changes in fiber length and CSA of fast fibers in the abdominal expiratory muscles reflect responses to chronic alterations in the mechanics of breathing that may affect muscle load, length, or the pattern of activity.     

Inhibition of muscle-specific gene expression by Id3: requirement of the C-terminal region of the protein for stable expression and function.

We have examined the role of an Id-like protein, Id3 (also known as HLH462), in the regulation of muscle-specific gene expression. Id proteins are believed to block expression of muscle-specific genes by preventing the dimerization between ubiquitous bHLH proteins (E proteins) and myogenic bHLH proteins such as MyoD. Consistent with its putative role as an inhibitor of differentiation, Id3 mRNA was detected in proliferating skeletal muscle cells, was further induced by basic fibroblast growth factor (bFGF) and was down-regulated in differentiated muscle cultures. Overexpression of Id3 efficiently inhibited the MyoD-mediated activation of the muscle-specific creatine kinase (MCK) reporter gene. Deletion analysis indicated that the C-terminal 15 amino acids of Id3 are critical for the full inhibitory activity while deleting up to 42 residues from the C-terminus of the related protein, Id2, did not affect its ability to inhibit the MCK reporter gene. Chimeric protein containing the N-terminal region of Id3 and the C-terminus of Id2 was also non-functional in transfected cells. In contrast, wild-type Id3, the C-terminal mutants, and the Id3/Id2 chimera could all interact with the E-protein E47in vitro. Additional studies indicated that truncation of the Id3 C-terminus might have adversely affected the expression level of the mutant proteins but the Id3/Id2 chimera was stably expressed. Taken together, our results revealed a more complex requirement for the expression and proper function of the Id family proteins than was hitherto expected.     

Differential expression of two MyoD genes in fast and slow muscles of gilthead seabream ( Sparus aurata)

Members of the myogenic regulatory gene family, including MyoD, Myf5, Myogenin and MRF4, are specifically expressed in myoblast and skeletal muscle cells and play important roles in regulating skeletal muscle development and growth. They are capable of converting a variety of non-muscle cells into myoblasts and myotubes. To better understand their roles in the development of fish muscles, we have isolated the MyoD genomic genes from gilthead seabream (Sparus aurata), analyzed the genomic structures, patterns of expression and the regulation of muscle-specific expression. We have demonstrated that seabream contain two distinct non-allelic MyoDgenes, MyoD1 and MyoD2. Sequence analysis revealed that these two MyoD genes shared a similar gene structure. Expression studies demonstrated that they exhibited overlapping but distinct patterns of expression in seabream embryos and adult slow and fast muscles. MyoD1 was expressed in adaxial cells that give rise to slow muscles, and lateral somitic cells that give rise to fast muscles. Similarly, MyoD2 was initially expressed in both slow and fast muscle precursors. However, MyoD2 expression gradually disappeared in the adaxial cells of 10- to 15-somite-stage embryos, whereas its expression in fast muscle precursor cells was maintained. In adult skeletal muscles, MyoD1 was expressed in both slow and fast muscles, whereas MyoD2 was specifically expressed in fast muscles. Treating seabream embryos with forskolin, a protein kinase A activator, inhibited MyoD1 expression in adaxial cells, while expression in fast muscle precursors was not affected. Promoter analysis demonstrated that both MyoD1 and MyoD2 promoters could drive green fluorescence protein expression in muscle cells of zebrafish embryos. Together, these data suggest that the two non-allelic MyoD genes are functional in seabream and their expression is regulated differently in fast and slow muscles. Hedgehog signaling is required for induction of MyoDexpression in adaxial cells.