Expression of stress proteins and mitochondrial chaperonins in chronically stimulated skeletal muscle (original) (raw)
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Heat shock protein 60 (Hsp60) is a chaperone localizing in skeletal muscle mitochondria, whose role is poorly understood. In the present study, the levels of Hsp60 in fibres of the entire posterior group of hindlimb muscles (gastrocnemius, soleus, and plantaris) were evaluated in mice after completing a 6-week endurance training program. The correlation between Hsp60 levels and the expression of four isoforms of peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC1α) were investigated only in soleus. Short-term overexpression of hsp60, achieved by in vitro plasmid transfection, was then performed to determine whether this chaperone could have a role in the activation of the expression levels of PGC1α isoforms. The levels of Hsp60 protein were fibre-type specific in the posterior muscles and endurance training increased its content in type I muscle fibers. Concomitantly with the increased levels of Hsp60 released in the blood stream of trained mice, mitochondrial copy number and the expression of three isoforms of PGC1α increased. Overexpressing hsp60 in cultured myoblasts induced only the expression of PGC1 1α, suggesting a correlation between Hsp60 overexpression and PGC1 1 α activation. The chaperoning system participates in many cellular functions from assisting protein folding and assembling of multimolecular complexes to maintaining the correct shape of enzymes 1–7. In addition, extracellular chaperones contribute to the intercommunication between different cells, tissues, and organs 8–10. It follows that exercise, which requires the participation of various intercommunicating muscle-cell types and intracellular components is most likely dependent on the chaperoning system, or at least some of its components. This is suggested by the presence of various heat shock protein (Hsp)-chaperones in skeletal muscle: sHsp, Hsp60, Hsp70, and Hsp90 11,12. A major deficiency in our knowledge of the chaperoning system pertains to its physiological distribution in the body and the changes that might occur during functional stages of the various organs. Elucidation of the chaperoning system components distinctive of each cell type in a tissue is necessary to fully understand its physiology and to identify pathological alterations that might be amenable to treatment targeting chaperones. Accordingly, we set out to map Hsp60 in skeletal muscle. We focused on Hsp60 because of its preeminent role in the physiology of mitochondria and the importance of the latter in muscle function and motor activities, and because it has been found extracellularly and in circulation, possibly intercommunicating cells. The three main
Mitochondria in the middle: Exercise preconditioning protection of striated muscle
The Journal of physiology, 2016
Cellular and physiological adaptations to an atmosphere which became enriched in molecular oxygen spurred the development of a layered system of stress protection, including antioxidant and stress response proteins. At physiological levels reactive oxygen and nitrogen species regulate cell signaling as well as intracellular and intercellular communication. Exercise and physical activity confer a variety of stressors on skeletal muscle and the cardiovascular system: mechanical, metabolic, oxidative. Transient increases of stressors during acute bouts of exercise or exercise training stimulate enhancement of cellular stress protection against future insults of oxidative, metabolic, and mechanical stressors that could induce injury or disease. This phenomenon has been termed both hormesis and exercise pre-conditioning (EPC). EPC stimulates transcription factors such as HIF-1, Nrf-1, HSF-1 and upregulates gene expression of a cadre of cytosolic (e.g. glutathione peroxidase, heat shock p...
Hsp78 chaperone functions in restoration of mitochondrial network following heat stress
Biochimica Et Biophysica Acta-molecular Cell Research, 2006
Under physiological conditions mitochondria of yeast Saccharomyces cerevisiae form a branched tubular network, the continuity of which is maintained by balanced membrane fusion and fission processes. Here, we show using mitochondrial matrix targeted green fluorescent protein that exposure of cells to extreme heat shock led to dramatic changes in mitochondrial morphology, as tubular network disintegrated into several fragmented vesicles. Interestingly, this fragmentation did not affect mitochondrial ability to maintain the membrane potential. Cells subjected to recovery at physiological temperature were able to restore the mitochondrial network, as long as an active matrix chaperone, Hsp78, was present. Deletion of HSP78 gene did not affect fragmentation of mitochondria upon heat stress, but significantly inhibited ability to restore mitochondrial network. Changes of mitochondrial morphology correlated with aggregation of mitochondrial proteins. On the other hand, recovery of mitochondrial network correlated with disappearance of protein aggregates and reactivation of enzymatic activity of a model thermo-sensitive protein: mitochondrial DNA polymerase. Since protein disaggregation and refolding is mediated by Hsp78 chaperone collaborating with Hsp70 chaperone system, we postulate that effect of Hsp78 on mitochondrial morphology upon recovery after heat shock is mediated by its ability to restore activity of unknown protein(s) responsible for maintenance of mitochondrial morphology.
Expression of heat shock protein 72 in atrophied rat skeletal muscles
Acta Physiologica Scandinavica, 2001
Changes in the expression of heat shock protein 72 (HSP72) in response to atrophic-inducing perturbations of muscle involving chronic mechanical unloading and denervation were determined. Adult male Wistar rats were assigned randomly to a sedentary cage control (CON), hind limb unloading (HU, via tail suspension), HU plus tenotomy (HU + TEN), HU plus denervation (HU + DEN), or HU + TEN + DEN group. Tenotomy and DEN involved cutting the Achilles tendon and removing a segment of the sciatic nerve, respectively. After 5 days, HSP72 levels in the soleus of the HU + DEN and HU + TEN + DEN groups were 42 (P < 0.05) and 53% (P < 0.01) less than CON, respectively. Soleus weight decreased in both groups. Heat shock protein 72 levels in the plantaris of the HU + TEN, HU + DEN, and HU + TEN + DEN groups were 31, 25, and 30% lower than CON, respectively (P < 0.05). Plantaris weight decreased in the HU + DEN and HU + TEN + DEN, but not in the HU + TEN group. Hind limb unloading alone had little effect on the HSP72 level in either muscle. Reduced levels of HSP72 were associated with a decreased soleus (r 0.62, P < 0.01) and plantaris (r 0.78, P < 0.001) weight. These results indicate that the levels of HSP72 in both a slow and a fast rat plantar¯exor are responsive to a chronic decrease in the levels of loading and/or activation and suggest that the neuromuscular activity level and the presence of innervation of a muscle are important factors that induce HSP72 expression.
Induction of heat shock protein 72 mRNA in skeletal muscle by exercise and training
Equine Veterinary Journal, 2010
In response to stress, cells synthesise heat shock proteins (HSP) to maintain protein homeostasis. To study whether exercise and training induce expression of HSP72 in the middle gluteal muscle, 10 Finnhorses performed a submaximal 60 min exercise test on a treadmill. Test A was performed after 3 months of training, and the other two tests 2 (B) and 5 (C) weeks later. Blood samples were taken during and after the tests, and biopsy samples before, immediately after and 23 h after each test. HSP72 mRNA was analysed using a digoxigenin-labelled probe. Blood lactate concentration in the 3 tests varied between 7.2 and 10.2 mmol/l. Training increased HSP72 mRNA, as indicated by increases in samples taken at rest (A<B<C). Exercise also tended to increase HSP72 mRNA transiently but, 23 h later, values had returned to pre-exercise levels. HSP72 mRNA was expressed in all muscle fibres. After exercise, HSP72 mRNA correlated positively with the peak concentration of blood lactate, but not with indicators of energy status. Therefore, acidosis rather than energy depletion was the major inducer of HSP72 expression after moderate intensity exercise. Because HSP72 may protect cells against stress, knowledge about their expression may help in planning optimal trainng regimes.
FEBS Letters, 1994
We have isolated a cDNA clone encoding chaperonin 10 from rat liver. The cDNA specifies a protein of 102 amino acids which, when transcribed and translated in vitro, yields a single basic product (PI > 9) that co-migrates exactly with the heat shock inducible cpnl0 of rat hepatoma cells during 2D gel-electrophoresis. It is concluded that cpnl0, unlike the majority of nuclear-encoded proteins of the mitochondrial matrix, is synthesised without a cleavable targeting signal and that, following removal of the initiating methionine, it becomes acetylated prior to mitochondrial import. Incubation of 'H-or ?+labelled cpnl0 with mitochondria confirms these conclusions and shows that cpnl0 is imported into mitochondria in an energy-dependent process which is inhibited by the presence of 2,4_dinitrophenol.
American Journal of Physiology-regulatory Integrative and Comparative Physiology, 2008
The purpose of the present study was to 1) develop a stable model for measuring contraction-induced elevations in mRNA in single skeletal muscle fibers; and 2) to utilize this model to investigate the response of heat shock protein 72 (HSP72) mRNA following an acute bout of fatiguing contractions. Living, intact skeletal muscle fibers were microdissected from lumbrical muscle of Xenopus laevis and either electrically stimulated for 15 min of tetanic contractions (EX; n=26) or not stimulated to contract (REST; n=14). The relative mean developed tension of EX fibers decreased to 29+7% of initial peak tension at the stimulation end-point. Following treatment, individual fibers were allowed to recover for 1 hr (n=9), 2 hr (n=8) or 4 hr (n=9) prior to isolation of total cellular mRNA. HSP72, heat shock protein 60 (HSP60) and cardiac -actin mRNA content were then assessed in individual fibers using qPCR detection. Relative HSP72 mRNA content was significantly (p<0.05) elevated at the 2 hr post-contraction time point relative to REST fibers when normalized to either HSP60 (18.5+7.5 fold) or cardiacactin (14.7+4.3 fold), although not at the 1 hr or 4 hr time points. These data indicate that: 1) extraction of RNA followed by relative quantification of mRNA of select genes in isolated single skeletal muscle fibers can be reliably performed; 2) HSP60 and cardiac -actin are suitable endogenous normalizing genes in skeletal muscle following contractions; 3) a significantly elevated content of HSP72 mRNA is detectable in skeletal muscle 2 hours after a single bout of fatiguing contractions despite minimal temperature changes and without influence from extracellular sources. .
Selective Induction of Mitochondrial Chaperones in Response to Loss of the Mitochondrial Genome
European Journal of Biochemistry, 1996
Molecular chaperones are known to play key roles in the synthesis, transport and folding of nuclearencoded mitochondrial proteins and of proteins encoded by mitochondrial DNA. Although the regulation of heat-shock genes has been the subject of considerable investigation, regulation of the genes encoding mitochondrial chaperones is not well defined. We have found that stress applied specifically to the mitochondria of mammalian cells is capable of eliciting an organelle-specific, molecular chaperone response. Using the loss of mitochondrial DNA as a means of producing a specific mitochondrial stress, we show by Western-blot analysis that mtDNA-less (e") rat hepatoma cells show an increase in the steady-state levels of chaperonin 60 (cpn 60) and chaperonin 10 (cpn 10). Nuclear transcription assays show that the upregulation of these chaperones is due to transcriptional activation. There was no effect on the inducible cytosolic Hsp 70, Hsp 72, nor on mtHsp 70 in @' ) cells, leading us to concluded that stress applied selectively to mitochondria elicits a specific molecular chaperone response. Heat stress was able to provide an additional induction of cpn 60 and cpn 10 above that obtained for the @ state alone, indicating that these genes have separate regulatory elements for the specific mitochondrial and general stress responses. Since the mitochondrial-specific chaperones are encoded by nuclear DNA, there must be a mechanism for molecular communication between the mitochondrion and nucleus and this system can address how stress is communicated between these organelles. Fux: +61 3 9479 2467. Abbreviations. mtHsp 70, mitochondrial matrix located homologue of Hsp 70; cpn 60 and 10, chaperonins 60 and 10, respectively; PhMe-SOzF, phenylmethanesulphonyl fluoride; EtBr, ethidium bromide.
Muscles
The skeletal muscle is a highly plastic tissue that shows a remarkable adaptive capacity in response to acute and resistance exercise, and modifies its composition to adapt to use and disuse, a process referred to as muscle plasticity. Heat shock proteins (HSPs), a class of evolutionarily conserved molecular chaperones, have been implicated in the regulation of skeletal muscle plasticity. Here, we summarize key findings supporting the notion that HSPs are important components required to maintain skeletal muscle integrity and functionality. HSPs participate in the transcriptional program required for myogenesis and are activated following muscle exercise and injury. Their dysfunction, either as a consequence of improper expression or genetic mutations, contributes to muscle atrophy and leads to the development of myopathies and peripheral motor neuropathies. Denervation/reinnervation and repeated rounds of nerve degeneration/regeneration have been observed in motor neuropathies, sug...
Resistance training increases heat shock protein levels in skeletal muscle of young and old rats
Experimental Gerontology, 2006
Heat shock proteins (HSP) HSP72, HSC70 and HSP25 protein levels and mRNA levels of HSP72 genes (Hsp72K1, Hsp72-2, Hsp72K3) and HSC70 were examined in tibialis anterior muscles from young and old rats following 4.5 weeks of heavy resistance exercise. Young (3 months) (nZ10) and old (30 months) (nZ9) rats were subjected to 14 sessions of electrically evoked resistance training using stretch-shortening contractions of the left limb that activated the dorsiflexor muscle group, including the tibialis anterior muscle, while the right side served as the intra-animal control. Muscle wet weight of the left tibialis anterior increased by 15.6% in young animals compared to the untrained right side, while the aged rats demonstrated no significant hypertrophy based on muscle wet weight. There were no differences in mRNA expression between the control and experimental muscles in either the old or the young animals for any of the four genes examined. On the other hand, HSP72 levels as determined by Western blots were significantly (p!0.01) higher (968.8 and 409.1%) in the trained as compared to the contralateral control muscle in young and old animals, respectively. HSP25 expression was increased significantly (p!0.01) by training in muscles of young rats (943.1%) and old rats (420.3%). Moreover, there was no training by age interaction for HSP72, while a significant age and training by age effects were found in muscles for HSP25. There was no change in HSC70 protein expression in response to the training intervention in either age group. SOD-1 enzyme level increased by 66.6% in the trained muscles of the young rats, while this enzyme was 33% lower in trained muscles compared to the untrained control side in old rats. Moreover, a significant (p!0.05) training by age interaction was found for SOD-1 enzyme levels. This study suggests that fast contracting muscles in young and old animals are capable of increasing HSP expression in response to high intensity contractile stress. Furthermore, the data are consistent with the hypothesis that higher levels of oxidative stress in muscles of old animals limit HSP levels and/or function in response to high intensity contractile stress. q