Contractile activity-induced transcriptional activation of cytochrome C involves Sp1 and is proportional to mitochondrial ATP synthesis in C2C12 muscle cells.

Contractile activity induces adaptations in the expression of genes encoding skeletal muscle mitochondrial proteins; however, the putative signals responsible for these adaptations remain unknown. We used electrical stimulation (5 Hz, 65 V) of C2C12 muscle cells in culture to define some of the mechanisms involved in contractile activity-induced changes in cytochrome c gene expression. Chronic contractile activity (4 days, 3 h/day) augmented cytochrome c mRNA by 1.6-fold above control cells. This was likely mediated by increases in transcriptional activation, because cells transfected with full-length (-726 base pairs) or minimal (-66 base pairs) cytochrome c promoter/chloramphenicol acetyltransferase reporter constructs demonstrated contractile activity-induced 1.5-1.7-fold increases in the absence of contractile activity-induced increases in mRNA stability. Transcriptional activation of the -726 promoter was abolished when muscle contraction was inhibited at various subcellular locations by pretreatment with either the Na(+) channel blocker tetrodotoxin, the intracellular Ca(2+) chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester, or the myosin ATPase inhibitor 2,3-butanedione monoxime. It was further reduced in unstimulated cells when mitochondrial ATP synthesis was impaired using the uncoupler 2,4-dinitrophenol. Because the contractile activity-induced response was evident within the minimal promoter, electromobility shift assays performed within the first intron (+75 to +104 base pairs) containing Sp1 sites revealed an elevated DNA binding in response to contractile activity. This was paralleled by increases in Sp1 protein levels. Sp1 overexpression studies also led to increases in cytochrome c transactivation and mRNA levels. These data suggest that variations in the rate of mitochondrial ATP synthesis are important in determining cytochrome c gene expression in muscle cells and that this is mediated, in part, by Sp1-induced increases in cytochrome c transcription.

Invited Review: contractile activity-induced mitochondrial biogenesis in skeletal muscle.

Chronic contractile activity produces mitochondrial biogenesis in muscle. This adaptation results in a significant shift in adenine nucleotide metabolism, with attendant improvements in fatigue resistance. The vast majority of mitochondrial proteins are derived from the nuclear genome, necessitating the transcription of genes, the translation of mRNA into protein, the targeting of the protein to a mitochondrial compartment via the import machinery, and the assembly of multisubunit enzyme complexes in the respiratory chain or matrix. Putative signals involved in initiating this pathway of gene expression in response to contractile activity likely arise from combinations of accelerations in ATP turnover or imbalances between mitochondrial ATP synthesis and cellular ATP demand, and Ca(2+) fluxes. These rapid events are followed by the activation of exercise-responsive kinases, which phosphorylate proteins such as transcription factors, which subsequently bind to upstream regulatory regions in DNA, to alter transcription rates. Contractile activity increases the mRNA levels of nuclear-encoded proteins such as cytochrome c and mitochondrial transcription factor A (Tfam) and mRNA levels of upstream transcription factors like c-jun and nuclear respiratory factor-1 (NRF-1). mRNA level changes are often most evident during the postexercise recovery period, and they can occur as a result of contractile activity-induced increases in transcription or mRNA stability. Tfam is imported into mitochondria and controls the expression of mitochondrial DNA (mtDNA). mtDNA contributes only 13 protein products to the respiratory chain, but they are vital for electron transport and ATP synthesis. Contractile activity increases Tfam expression and accelerates its import into mitochondria, resulting in increased mtDNA transcription and replication. The result of this coordinated expression of the nuclear and the mitochondrial genomes, along with poorly understood changes in phospholipid synthesis, is an expansion of the muscle mitochondrial reticulum. Further understanding of 1) regulation of mtDNA expression, 2) upstream activators of NRF-1 and other transcription factors, 3) the identity of mRNA stabilizing proteins, and 4) potential of contractile activity-induced changes in apoptotic signals are warranted.

Effects of contractile activity on mitochondrial transcription factor A expression in skeletal muscle.

Mitochondrial transcription factor A (Tfam) is a nuclear-encoded gene product that is imported into mitochondria and is required for the transcription of mitochondrial DNA (mtDNA). We hypothesized that conditions known to produce mitochondrial biogenesis in skeletal muscle would be preceded by an increase in Tfam expression. Therefore, rat muscle was stimulated (10 Hz, 3 h/day). Tfam mRNA levels were significantly elevated (by 55%) at 4 days and returned to control levels at 14 days. Tfam import into intermyofibrillar (IMF) mitochondria was increased by 52 and 61% (P < 0.05) at 5 and 7 days, respectively. This corresponded to an increase in the level of import machinery components. Immunoblotting data indicated that IMF Tfam protein content was increased by 63% (P < 0.05) at 7 days of stimulation. This was associated with a 49% (P < 0.05) increase in complex formation at the mtDNA promoter and a 65% (P < 0.05) increase in the levels of a mitochondrial transcript, cytochrome-c oxidase (COX) subunit III. Similarly, COX enzyme activity was elevated by 71% (P < 0.05) after 7 days of contractile activity. These results indicate that early events in mitochondrial biogenesis include increases in Tfam mRNA, followed by accelerations in mitochondrial import and increased Tfam content, which correspond with increased binding to the mtDNA promoter region. This was accompanied by increased mitochondrial transcript levels and elevated COX activity. These data support the role of Tfam as a regulatory protein involved in contractile activity-induced mitochondrial biogenesis.

Tom20-mediated mitochondrial protein import in muscle cells during differentiation.

Mitochondrial biogenesis is accompanied by an increased expression of components of the protein import machinery, as well as increased import of proteins destined for the matrix. We evaluated the role of the outer membrane receptor Tom20 by varying its expression and measuring changes in the import of malate dehydrogenase (MDH) in differentiating C2C12 muscle cells. Cells transfected with Tom20 had levels that were twofold higher than in control cells. Labeling of cells followed by immunoprecipitation of MDH revealed equivalent increases in MDH import. This parallelism between import rate and Tom20 levels was also evident as a result of thyroid hormone treatment. Using antisense oligodeoxynucleotides, we inhibited Tom20 expression by 40%, resulting in 40-60% reductions in MDH import. In vitro assays also revealed that import into the matrix was more sensitive to Tom20 inhibition than import into the outer membrane. These data indicate a close relationship between induced changes in Tom20 and the import of a matrix protein, suggesting that Tom20 is involved in determining the kinetics of import. However, this relationship was dissociated during normal differentiation, since the expression of Tom20 remained relatively constant, whereas imported MDH increased 12-fold. Thus Tom20 is important in determining import during organelle biogenesis, but other mechanisms (e.g., intramitochondrial protein degradation or nuclear transcription) likely also play a role in establishing the final mitochondrial phenotype during normal muscle differentiation.

Assembly of the cellular powerhouse: current issues in muscle mitochondrial biogenesis.

Mitochondrial biogenesis occurs in muscle in response to chronic exercise, resulting in fatigue resistance. The assembly of the organelle is initiated by contraction-induced signals, which lead to the transcriptional activation of nuclear genes. This is accompanied by alterations in mRNA stability, as well as increases in protein import and mitochondrial DNA copy number, leading to a greater muscle mitochondrial content.

Effect of contractile activity on protein turnover in skeletal muscle mitochondrial subfractions.

To determine the role of intramitochondrial protein synthesis (PS) and degradation (PD) in contractile activity-induced mitochondrial biogenesis, we evaluated rates of [(35)S]methionine incorporation into protein in isolated rat muscle subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondria. Rates of PS ranged from 47 to 125% greater (P < 0.05) in IMF compared with SS mitochondria. Intense, acute in situ contractile activity (10 Hz, 5 min) of fast-twitch gastrocnemius muscle resulted in a 50% decrease in PS (P < 0.05) in SS but not IMF mitochondria. Recovery, or continued contractile activity (55 min), reestablished PS in SS mitochondria. In contrast, PS was not affected in either SS or IMF mitochondria after prolonged (60-min) contractile activity in the presence or absence of a recovery period. PD was not influenced by 5 min of contractile activity in the presence or absence of recovery but was reduced after 60 min of contractions followed by recovery. Chronic stimulation (10 Hz, 3 h/day, 14 days) increased muscle cytochrome-c oxidase activity by 2.2-fold but reduced PS in IMF mitochondria by 29% (P < 0.05; n = 4). PS in SS mitochondria and PD in both subfractions were not changed by chronic stimulation. Thus acute contractile activity exerts differential effects on protein turnover in IMF and SS mitochondria, and it appears that intramitochondrial PS does not limit the extent of chronic contractile activity-induced mitochondrial biogenesis.

Effect of thyroid hormone on mtHsp70 expression, mitochondrial import and processing in cardiac muscle.

Mitochondrial heat shock protein 70 (mtHsp70), an important mitochondrial chaperone, is increased in cardiac muscle mitochondria of hyperthyroid rats. To determine the mechanism(s) underlying this increase, we used variations in thyroid status. In Series I, rats were made hyperthyroid by injecting them with 3,3', 5-triiodo-l-thyronine (T(3)) for 5 days, or by treating them with vehicle. In Series II, animals were given 6-n-propyl-2-thiouracil in their drinking water (0.05% w/v) for a period of 32-42 days to make them hypothyroid. During the last 5 days of treatment these animals received injections of either T(3) or vehicle. T(3) treatment resulted in parallel increases in mtHsp70 protein and mRNA levels in a variety of tissues, suggesting transcriptional regulation. However, evidence of tissue-specific post-transcriptional regulation was also apparent. In isolated heart mitochondria, T(3) treatment resulted in a 1.8-fold increase in mtHsp70. This was due to the 1. 6-fold greater import of mtHsp70 into mitochondria in T(3), compared with hypothyroid animals, and it could not be attributed to an altered rate of intramitochondrial mtHsp70 degradation. The rate of processing of mtHsp70 to its mature form, reflecting mitochondrial processing peptidase activity, was unaffected by T(3), but was more rapid than mtHsp70 import. These data indicate a novel mechanism by which T(3) modifies the mitochondrial phenotype via the adaptations in the protein import pathway.

Zidovudine (AZT) induced alterations in mitochondrial biogenesis in rat striated muscles.

Zidovudine (AZT) and didanosine (ddI), two drugs used in the treatment of AIDS, are also known to cause mitochondrial abnormalities. We investigated the physiological relevance of the mitochondrial defects by measuring in situ skeletal muscle performance and cytochrome c oxidase (CYTOX) enzyme activity in heart muscle, red highoxidative (RG) and white low-oxidative (WG) portions of the gastrocnemius muscle of control (n = 17), AZT-(n = 14), or ddI-treated (n = 11) rats for 28 days. We also evaluated the hypothesis that AZT treatment could alter the expression of the mitochondrial transcription factor A (mtTFA), a key molecule involved in mitochondrial DNA (mtDNA) replication and transcription. AZT had a pronounced effect on blood pressure and skeletal muscle performance, which were significantly decreased during contractile activity at 2 and 5 Hz, compared with control. A significant decrease in CYTOX activity in heart and RG, but not WG muscles, was also evident. In the heart, this was accompanied by an apparent compensatory increase in mtTFA mRNA level that could not be attributed to enhanced transcriptional activation mediated by nuclear respiratory factor 1 (NRF-1). In contrast with AZT, no effect of ddI was found on the extent of fatigue or muscle enzyme activity. These results indicate that AZT induces mitochondrial defects primarily in muscles with the highest oxidative capacities (heart and RG). The long-term effects of AZT on mitochondrial biogenesis have the potential to reduce muscle performance, but the effects on performance in this short-term study were likely due to an inability of the AZT-treated animals to maintain blood pressure during contractile activity.

Cytochrome c transcriptional activation and mRNA stability during contractile activity in skeletal muscle.

We evaluated contractile activity-induced alterations in cytochrome c transcriptional activation and mRNA stability with unilateral chronic stimulation (10 Hz, 3 h/day) of the rat tibialis anterior (TA) muscle for 1, 2, 3, 4, 5, and 7 days (n = 3-11/group). Transcriptional activation was assessed by direct plasmid DNA injection into the TA with a chloramphenicol acetyltransferase (CAT) reporter gene linked to 326 bp of the cytochrome c promoter. Cytochrome c mRNA in stimulated muscles increased by 1.3- to 1. 7-fold above control between 1 and 7 days. Cytochrome c protein was increased after 5 days of stimulation to reach levels that were 1. 9-fold higher than control by 7 days. Cytochrome c mRNA stability, determined with an in vitro decay assay, was greater in stimulated TA than in control between 2 and 4 days, likely mediated by the induction of a cytosolic factor. In contrast, cytochrome c transcriptional activation was elevated only after 5 days of stimulation when mRNA stability had returned to control levels. Thus the contractile activity-induced increase in cytochrome c mRNA was due to an early increase in mRNA stability, followed by an elevation in transcriptional activation, leading to an eventual increase in cytochrome c protein levels.

Calcium-dependent regulation of cytochrome c gene expression in skeletal muscle cells. Identification of a protein kinase c-dependent pathway.

Mitochondrial biogenesis can occur rapidly in mammalian skeletal muscle subjected to a variety of physiological conditions. However, the intracellular signal(s) involved in regulating this process remain unknown. Using nuclearly encoded cytochrome c, we show that its expression in muscle cells is increased by changes in cytosolic Ca2+ using the ionophore A23187. Treatment of myotubes with A23187 increased cytochrome c mRNA expression up to 1.7-fold. Transfection experiments using promoter-chloramphenicol acetyltransferase constructs revealed that this increase could be transcriptionally mediated since A23187 increased chloramphenicol acetyltransferase activity by 2.5-fold. This increase was not changed by KN62, an inhibitor of Ca2+/calmodulin-dependent kinases II and IV, and it was not modified by overexpression of protein kinase A and cAMP response element-binding protein, demonstrating that the A23187 effect was not mediated through Ca2+/calmodulin-dependent kinase- or protein kinase A-dependent pathways. However, treatment of myotubes with staurosporine or 12-O-tetradecanoylphorbol-13-acetate reduced the effect of A23187 on cytochrome c transactivation by 40-50%. Coexpression of the Ca2+-sensitive protein kinase C isoforms alpha and betaII, but not the Ca2+-insensitive delta isoform, exaggerated the A23187-mediated response. The short-term effect of A23187 was mediated in part by mitogen-activated protein kinase (extracellular signal-regulated kinases 1 and 2) since its activation peaked 2 h after A23187 treatment, and cytochrome c transactivation was reduced by PD98089, a mitogen-activated protein kinase/extracellular signal-regulated kinase kinase inhibitor. These results demonstrate the existence of a Ca2+-sensitive, protein kinase C-dependent pathway involved in cytochrome c expression and implicate Ca2+ as a signal in the up-regulation of nuclear genes encoding mitochondrial proteins.

Thyroid hormone modifies mitochondrial phenotype by increasing protein import without altering degradation.

The mitochondrial phenotype within cardiac muscle cells is dramatically altered by thyroid hormone. We report here that this can be accounted for, in part, by modifications in the rate of mitochondrial protein import. The import of matrix-localized precursor proteins malate dehydrogenase (MDH) and ornithine carbamoyltransferase was augmented, whereas the insertion of the outer membrane protein Bcl-2 was unaffected by thyroid hormone treatment. Coincident with increases in the import of these matrix-localized precursors were thyroid hormone-induced elevations in the outer membrane receptor Tom20 and the matrix heat-shock protein mthsp70. The phospholipid cardiolipin was not involved in mediating the thyroid hormone-induced increase in import, as judged from adriamycin inhibition studies. When the import reaction was supplemented with rat heart cytosol, we found that 1) MDH import was stimulated, but Bcl-2 import was inhibited and 2) thyroid hormone did not influence the effect of the cytosol on import rates. Thus distinct requirements exist for the mitochondrial import of precursor proteins, destined for different organellar compartments. Although import of these matrix-localized proteins was augmented by thyroid hormone treatment, the proteolysis of matrix proteins was unaffected as indicated by the degradation of cytob2(167)RIC-dihydrofolate reductase, a chimeric protein missorted to the matrix. Thus our data indicate that at least some thyroid hormone-induced modifications of the mitochondrial phenotype occur due to the compartment-specific upregulation of precursor protein import rates, likely mediated via changes in the expression of protein import machinery components.

Contractile activity-induced adaptations in the mitochondrial protein import system.

We previously demonstrated that subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondrial subfractions import proteins at different rates. This study was undertaken to investigate 1) whether protein import is altered by chronic contractile activity, which induces mitochondrial biogenesis, and 2) whether these two subfractions adapt similarly. Using electrical stimulation (10 Hz, 3 h/day for 7 and 14 days) to induce contractile activity, we observed that malate dehydrogenase import into the matrix of the SS and IMF mitochondia isolated from stimulated muscle was significantly increased by 1.4-to 1.7-fold, although the pattern of increase differed for each subfraction. This acceleration of import may be mitochondrial compartment specific, since the import of Bcl-2 into the outer membrane was not affected. Contractile activity also modified the mitochondrial content of proteins comprising the import machinery, as evident from increases in the levels of the intramitochondrial chaperone mtHSP70 as well as the outer membrane import receptor Tom20 in SS and IMF mitochondria. Addition of cytosol isolated from stimulated or control muscles to the import reaction resulted in similar twofold increases in the ability of mitochondria to import malate dehydrogenase, despite elevations in the concentration of mitochondrial import-stimulating factor within the cytosol of chronically stimulated muscle. These results suggest that chronic contractile activity modifies the extra- and intramitochondrial environments in a fashion that favors the acceleration of precursor protein import into the matrix of the organelle. This increase in protein import is likely an important adaptation in the overall process of mitochondrial biogenesis.

Effect of microgravity on the expression of mitochondrial enzymes in rat cardiac and skeletal muscles.

The purpose of this study was to examine the expression of nuclear and mitochondrial genes in cardiac and skeletal muscle (triceps brachii) in response to short-duration microgravity exposure. Six adult male rats were exposed to microgravity for 6 days and were compared with six ground-based control animals. We observed a significant 32% increase in heart malate dehydrogenase (MDH) enzyme activity, which was accompanied by a 62% elevation in heart MDH mRNA levels after microgravity exposure. Despite modest elevations in the mRNAs encoding subunits III, IV, and VIc as well as a 2.2-fold higher subunit IV protein content after exposure to microgravity, heart cytochrome c oxidase (CytOx) enzyme activity remained unchanged. In skeletal muscle, MDH expression was unaffected by microgravity, but CytOx activity was significantly reduced 41% by microgravity, whereas subunit III, IV, and VIc mRNA levels and subunit IV protein levels were unaltered. Thus tissue-specific (i.e., heart vs. skeletal muscle) differences exist in the regulation of nuclear-encoded mitochondrial proteins in response to microgravity. In addition, the expression of nuclear-encoded proteins such as CytOx subunit IV and expression of MDH are differentially regulated within a tissue. Our data also illustrate that the heart undergoes previously unidentified mitochondrial adaptations in response to short-term microgravity conditions more dramatic than those evident in skeletal muscle. Further studies evaluating the functional consequences of these adaptations in the heart, as well as those designed to measure protein turnover, are warranted in response to microgravity.

Influence of aging on protein import into cardiac mitochondria.

This study was undertaken to determine whether age-related changes in the content and composition of cardiac mitochondria could be due, in part, to alterations in mitochondrial protein import. Precursor proteins malate dehydrogenase and ornithine carbamoyltransferase were synthesized by in vitro transcription and translation and were incubated with mitochondria isolated from the hearts of young (4-mo), old (22-mo), and senescent (28-mo) rats. Mitochondria from senescent animals exhibited a twofold higher import rate of both precursors into the matrix compartment compared with mitochondria from young and old animals. The expression of glucose regulated protein 75 and heat shock protein 60, two matrix chaperonins that are essential for import, was elevated in the mitochondria of both old and senescent animals before the observed changes in import. Import was equally affected in senescent and young heart mitochondria by inhibition of cardiolipin, a mitochondrial phospholipid involved in protein translocation. The results indicate that the altered mitochondrial phenotype evident in the aging myocardium cannot be accounted for by reduced rates of protein import. Furthermore, levels of cardiolipin and matrix chaperonins do not appear to be rate-limiting steps in the import process. These data suggest that the protein import step of mitochondrial assembly is subject to adaptations under pathophysiological conditions.

Protein import into subsarcolemmal and intermyofibrillar skeletal muscle mitochondria. Differential import regulation in distinct subcellular regions.

To date, no studies have described the import of proteins in mitochondria obtained from skeletal muscle. In this tissue, mitochondria consist of the functionally and biochemically distinct intermyofibrillar (IMF) and subsarcolemmal (SS) subfractions, which are localized in specialized cellular compartments. This mitochondrial heterogeneity in muscle could be due, in part, to differential rates of protein import. To evaluate this possibility, the import of precursor malate dehydrogenase and ornithine carbamyltransferase proteins was investigated in isolated IMF and SS mitochondria in vitro. Import of these was 3-4-fold greater in IMF compared with SS mitochondria as a function of time. This could account for the higher malate dehydrogenase enzyme activity in IMF mitochondria. Divergent import rates in IMF and SS mitochondria likely result from a differential reliance on various components of the import pathway. SS mitochondria possess a greater content of the molecular chaperones hsp60 and Grp75, yet import is lower than in IMF mitochondria. On the other hand, adriamycin inhibition studies illustrated a greater reliance on acidic phospholipids (i.e. cardiolipin) for the import process in SS mitochondria. Matrix ATP levels were 3-fold higher in IMF mitochondria, but experiments in which ATP depletion was performed with atractyloside and oligomycin illustrated a dissociation between import rates and levels of ATP. In contrast, a close relationship was found between the rate of ATP production (i.e. mitochondrial respiration) and protein import. When respiratory rates in IMF and SS mitochondria were equalized, import rates in both subfractions were similar. These data indicate that 1) import rates are more closely related to the rate of ATP production than the steady state ATP level, 2) import into IMF and SS mitochondrial subfractions is regulated differently, and 3) mitochondrial heterogeneity within a cell type can be due to differences in the rates of protein import, suggesting that this step is a potentially regulatable event in determining the final mitochondrial phenotype.

Tissue-specific stability of nuclear- and mitochondrially encoded mRNAs.

Steady-state levels of mRNAs encoding mitochondrial proteins are drastically different among tissues. We evaluated tissue-specific variations in mRNA stability by comparing rates of mRNA decay in liver, heart, and muscle following the inhibition of transcription. Rates of decline of the mRNAs encoding delta-aminolevulinate synthase (ALAs), cytochrome c oxidase subunit VIc (nuclear-encoded), and subunit III (mitochondrially encoded) in heart, liver, and muscle for 6 h following transcription inhibition with actinomycin D or ethidium bromide were measured. Subunit VIc mRNA levels were least stable in liver (t1/2 = 2.4 h), slightly greater in heart (t1/2 = 3.3 h), and very stable in skeletal muscle. Similarly, ALAs mRNA exhibited a t1/2 of 41 min in liver, but this was markedly increased to approximately 11-14 h in heart and skeletal muscle. In contrast, subunit III was least stable in heart (t1/2 = 2.1 h), somewhat more stable in liver (t1/2 = 3.8 h), but no decline in subunit III mRNA levels occurred in muscle following the inhibition of transcription. Thus, muscle, heart, and liver possess tissue-specific mechanisms which control the stability of mRNAs encoding mitochondrial proteins. In addition, the coordinated expression of subunit III and VIc mRNAs is different tissues is partly due to parallel rates of mRNA turnover. This suggests the presence of intra- and extramitochondrial factors within a tissue which regulate the stability of specific mRNAs in a similar manner.

Dystrophin, vinculin, and aciculin in skeletal muscle subject to chronic use and disuse.

Dystrophin is a subsarcolemmal protein that interacts with cytoskeletal actin and a glycoprotein complex in the plasma membrane. One potential function of dystrophin is its ability to stabilize the sarcolemmal membrane during muscle contraction. We hypothesized 1) that chronic muscle use and disuse would alter the expression of dystrophin as a compensatory mechanism designed to prevent muscle damage, and 2) that other subsarcolemmal cytoskeletal proteins (vinculin, M-vinculin, aciculin 60/63 kDa) that colocalize with dystrophin in muscle adherens junctions would be changed in parallel. Chronic muscle use induced by voluntary running or 10-Hz chronic stimulation did not alter dystrophin levels in rat muscle. In contrast, muscle disuse induced by 6 d of microgravity, or 7 and 21 d of denervation, increased dystrophin levels by 1.8-, 1.9- and 3.2-fold, respectively. Thus, this increase in dystrophin levels appears to be dependent on the duration of muscle disuse, independent of the presence of the nerve. Denervation also induced 3.3-fold increases in vinculin and aciculin 60 kDa, in parallel with dystrophin. However, in contrast to its effects on dystrophin, chronic stimulation increased the levels of vinculin and aciculin 60 kDa by 3.4- and 6.4-fold, respectively. Thus, both the removal and the augmentation of muscle activity resulted in increases of these two cytoskeletal proteins. The data indicate that the concentrations of these proteins are independently regulated. They further indicate that chronic muscle use is not a stimulus for the induction of dystrophin levels, suggesting that normal levels are sufficient for the protective effect on the sarcolemma that dystrophin may confer. The results reveal an interesting area of muscle plasticity, and the adaptation observed may have profound implications for the structure and function of skeletal muscle responding to changes in contractile activity.

Effects of hypothyroidism and aortic construction on mitochondria during cardiac hypertrophy.

We evaluated mitochondrial adaptations in the hearts of euthyroid and hypothyroid rats subject to aortic constriction for 2, 4, 7, 14, 21, and 28 d to induce a pressure-overload (PO), compared to sham-operated (SH) controls. PO animals attained higher arterial pressures than SH animals, by 55% in the euthyroid group, but only 14% in hypothyroid rats after 28 d. The left ventricle/body weight ratio was increased 44% by PO in the euthyroid group, and 26% in the hypothyroid group. PO attenuated the decline in cardiac growth in the hypothyroid group. Thus, hypothyroidism reduces the magnitude of the PO, but not the potential for hypertrophy in response to PO. Cytochrome c oxidase activity (CYTOX) was unchanged by PO in the euthyroid animals, indicating that the synthesis of mitochondria paralleled adaptive growth. However, CYTOX activity decreased up to 20% in the hypothyroid groups (P < 0.05) and was unaltered by PO. Thus, PO prevented the decline in growth, but not the decline in mitochondrial enzymes due to hypothyroidism. The lack of effect of PO on mitochondria was partly due to pretranslational changes since CYTOX subunit VIc mRNA was reduced by PO in the hypothyroid animals, but not in the euthyroid group. Levels of the chaperones HSP60 and GRP75, as well as HSP60 mRNA were unaffected by hypothyroidism, but paralleled adaptive growth induced by PO. Hypothyroidism changes the pattern of gene expression within the heart leading to altered mitochondrial composition. This cannot be compensated for by conditions of increased physiological demand.

Expression of stress proteins and mitochondrial chaperonins in chronically stimulated skeletal muscle.

Molecular chaperones and cytosolic stress proteins are actively involved in the stabilization, import and refolding of precursor proteins into mitochondria. The purpose of the present study was to evaluate the relationship between mitochondrial content under steady-state conditions, and during the induction of organelle biogenesis, with the expression of stress proteins and mitochondrial chaperonins. A comparison of steady-state levels of mitochondrial enzyme activity [cytochrome c oxidase (CYTOX)] with chaperonin levels [the heat-shock protein HSP60, the glucose-regulated protein GRP75 (mtHSP70)] in striated muscles possessing a wide range of oxidative capacities revealed a proportional expression between the two. This relationship was disrupted by chronic contractile activity brought about by 10 days of 10 Hz stimulation of the tibialis anterior (TA) muscle, which induced 2.4-fold increases in CYTOX activity, but 3.2- and 9.3-fold increases in HSP60 and GRP75 respectively. The inducible stress protein HSP70i was detected at low levels in control TA muscle, and was increased 9.6-fold by chronic contractile activity, to values comparable with those found in the unstressed soleus muscle. This increase occurred in the absence of changes in type I MHC levels, indicating independent regulation of these genes. Despite the increases in HSP60 and HSP70i proteins, contractile activity did not alter their respective mRNA levels, illustrating post-transcriptional mechanisms of gene regulation during contractile activity. In contrast, the mRNA levels encoding the co-chaperonin CPN10 were increased 3.3-fold by contractile activity. Thus, the expression of individual mitochondrial chaperonins is independently regulated and uncoordinated. The extent of the induction of these stress proteins and chaperonins by contractile activity exceeded that of membrane enzymes (e.g. CYTOX). It remains to be determined whether this marked induction of proteins comprising part of the protein import machinery is beneficial for the translocation of enzyme precursors into the mitochondria during conditions of accelerated biogenesis.